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Choy EYW, Leong CO, Cheong SK, Then KL, Then KY. Adeno-Associated Virus-Engineered Umbilical Cord-Derived Mesenchymal Stromal Cells Overexpressing Human sFlt-1 for Anti-Angiogenesis. Life (Basel) 2025; 15:728. [PMID: 40430157 DOI: 10.3390/life15050728] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2025] [Revised: 03/31/2025] [Accepted: 04/09/2025] [Indexed: 05/29/2025] Open
Abstract
PURPOSE Genetic engineering of mesenchymal stromal cells (MSCs) using viral vectors has emerged as a promising approach to enhance the efficacy of anti-angiogenic gene therapies. Umbilical cord-derived MSCs are an attractive cell source due to their easy accessibility and potential for genetic modification. Adeno-associated viruses (AAVs) have been utilized in clinical settings to deliver therapeutic genes due to its characteristic of transient integration into the genome. In this study, we investigated the efficacy of using recombinant AAV-engineered umbilical cord-derived MSCs overexpressing anti-angiogenic factor, hsFlt-1 (MSCs.hsFlt1). METHODS The plasmid containing the hsFlt-1 gene was cloned into the AAV2 target backbone and validated using Sanger sequencing. The transduction process was studied to determine the optimal conditions, including the effect of MOI, media serum percentage, and attachment of MSCs, to achieve higher transduction efficiency. The functionality of MSCs.hsFtl1 was analyzed using qPCR, ELISA, and tube formation assays. RESULTS MSCs.hsFtl1 transduced at an MOI of 1 × 106 demonstrated high transduction efficiency and exhibited robust gene and protein expression of hsFlt-1. The results revealed significant inhibition of growth in human umbilical vein endothelial cells (HUVECs) using a remarkably low dose of MSCs.hsFlt1 at 12.3 ng/mL. This observed anti-angiogenic effect was comparable to the clinically used Bevacizumab. CONCLUSIONS The anti-angiogenic potential of MSCs.hsFlt1 effectively demonstrated in this study suggests their promising utility for targeted anti-angiogenic gene therapy approaches.
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Affiliation(s)
- Ewa Yee-Wa Choy
- School of Postgraduate Studies, Faculty of Medicine and Health, IMU University, Jalan Jalil Perkasa 19, Bukit Jalil, Kuala Lumpur 57000, Malaysia
- CryoCord Sdn Bhd, 1, Bio X Centre, Persiaran Cyber Point Selatan, Cyberjaya 63000, Selangor, Malaysia
| | - Chee-Onn Leong
- AGTC Genomics, J2-1, Pusat Perdagangan Bandar, Persiaran Jalil 1, Kuala Lumpur 57000, Malaysia
| | - Soon-Keng Cheong
- Hematology Department, National University of Malaysia (UKM), Jalan Ya'acob Latif, Bandar Tun Razak, Cheras, Kuala Lumpur 56000, Malaysia
- Department of Medicine, M. Kandiah Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Jalan Sungai Long, Bandar Sungai Long Cheras, Kajang 43000, Selangor, Malaysia
| | - Khong-Lek Then
- CryoCord Sdn Bhd, 1, Bio X Centre, Persiaran Cyber Point Selatan, Cyberjaya 63000, Selangor, Malaysia
| | - Kong-Yong Then
- CryoCord Sdn Bhd, 1, Bio X Centre, Persiaran Cyber Point Selatan, Cyberjaya 63000, Selangor, Malaysia
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2
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Calton MA, Croze RH, Burns C, Beliakoff G, Vazin T, Szymanski P, Schmitt C, Klein A, Leong M, Quezada M, Holt J, Bolender G, Barglow K, Khoday D, Mason T, Delaria K, Hassanipour M, Kotterman M, Khanani AM, Schaffer D, Francis P, Kirn D. Design and Characterization of a Novel Intravitreal Dual-Transgene Genetic Medicine for Neovascular Retinopathies. Invest Ophthalmol Vis Sci 2024; 65:1. [PMID: 39620832 PMCID: PMC11614000 DOI: 10.1167/iovs.65.14.1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2024] [Accepted: 10/28/2024] [Indexed: 12/06/2024] Open
Abstract
Purpose Intravitreal delivery of therapeutic transgenes to the retina via engineered viral vectors can provide sustained local concentrations of therapeutic proteins and thus potentially reduce the treatment burden and improve long-term vision outcomes for patients with neovascular (wet) age-related macular degeneration (AMD), diabetic macular edema (DME), and diabetic retinopathy. Methods We performed directed evolution in nonhuman primates (NHP) to invent an adeno-associated viral (AAV) variant (R100) with the capacity to cross vitreoretinal barriers and transduce all regions and layers of the retina following intravitreal injection. We then engineered 4D-150, an R100-based genetic medicine carrying 2 therapeutic transgenes: a codon-optimized sequence encoding aflibercept, a recombinant protein that inhibits VEGF-A, VEGF-B, and PlGF, and a microRNA sequence that inhibits expression of VEGF-C. Transduction, transgene expression, and biological activity were characterized in human retinal cells in vitro and in NHPs. Results R100 demonstrated superior retinal cell transduction in vitro and in vivo compared to AAV2, a commonly used wild-type AAV serotype in retinal gene therapies. Transduction of human retinal pigment epithelial cells in vitro by 4D-150 resulted in dose-dependent transgene expression and corresponding reductions in VEGF-A and VEGF-C. Intravitreal administration of 4D-150 to NHPs was well tolerated and led to robust retinal expression of both transgenes. In a primate model of laser-induced choroidal neovascularization, 4D-150 completely prevented clinically relevant angiogenic lesions at all tested doses. Conclusions These findings support further development of 4D-150. Clinical trials are underway to establish the safety and efficacy of 4D-150 in individuals with wet AMD and DME.
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Affiliation(s)
| | - Roxanne H. Croze
- 4D Molecular Therapeutics, Emeryville, California, United States
| | - Christian Burns
- 4D Molecular Therapeutics, Emeryville, California, United States
| | - Ghezal Beliakoff
- 4D Molecular Therapeutics, Emeryville, California, United States
| | - Tandis Vazin
- 4D Molecular Therapeutics, Emeryville, California, United States
| | - Paul Szymanski
- 4D Molecular Therapeutics, Emeryville, California, United States
| | | | - Austin Klein
- 4D Molecular Therapeutics, Emeryville, California, United States
| | - Meredith Leong
- 4D Molecular Therapeutics, Emeryville, California, United States
| | - Melissa Quezada
- 4D Molecular Therapeutics, Emeryville, California, United States
| | - Jenny Holt
- 4D Molecular Therapeutics, Emeryville, California, United States
| | - Gabe Bolender
- 4D Molecular Therapeutics, Emeryville, California, United States
| | | | - Devi Khoday
- 4D Molecular Therapeutics, Emeryville, California, United States
| | - Thomas Mason
- 4D Molecular Therapeutics, Emeryville, California, United States
| | | | | | | | - Arshad M. Khanani
- Sierra Eye Associates, Reno, Nevada, United States
- University of Nevada, Reno School of Medicine, Reno, Nevada, United States
| | - David Schaffer
- University of California, Berkeley, California, United States
| | - Peter Francis
- 4D Molecular Therapeutics, Emeryville, California, United States
| | - David Kirn
- 4D Molecular Therapeutics, Emeryville, California, United States
- University of California, Berkeley, California, United States
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3
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Giacomoni J, Åkerblom M, Habekost M, Fiorenzano A, Kajtez J, Davidsson M, Parmar M, Björklund T. Identification and validation of novel engineered AAV capsid variants targeting human glia. Front Neurosci 2024; 18:1435212. [PMID: 39193523 PMCID: PMC11348808 DOI: 10.3389/fnins.2024.1435212] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2024] [Accepted: 07/15/2024] [Indexed: 08/29/2024] Open
Abstract
Direct neural conversion of endogenous non-neuronal cells, such as resident glia, into therapeutic neurons has emerged as a promising strategy for brain repair, aiming to restore lost or damaged neurons. Proof-of-concept has been obtained from animal studies, yet these models do not efficiently recapitulate the complexity of the human brain, and further refinement is necessary before clinical translation becomes viable. One important aspect is the need to achieve efficient and precise targeting of human glial cells using non-integrating viral vectors that exhibit a high degree of cell type specificity. While various naturally occurring or engineered adeno-associated virus (AAV) serotypes have been utilized to transduce glia, efficient targeting of human glial cell types remains an unsolved challenge. In this study, we employ AAV capsid library engineering to find AAV capsids that selectively target human glia in vitro and in vivo. We have identified two families of AAV capsids that induce efficient targeting of human glia both in glial spheroids and after glial progenitor cell transplantation into the rat forebrain. Furthermore, we show the robustness of this targeting by transferring the capsid peptide from the parent AAV2 serotype onto the AAV9 serotype, which facilitates future scalability for the larger human brain.
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Affiliation(s)
- Jessica Giacomoni
- Developmental and Regenerative Neurobiology, Lund Stem Cell Center, Department of Experimental Medical Science, Faculty of Medicine, Lund University, Lund, Sweden
| | - Malin Åkerblom
- Molecular Neuromodulation, Department of Experimental Medical Science, Faculty of Medicine, Lund University, Lund, Sweden
| | - Mette Habekost
- Developmental and Regenerative Neurobiology, Lund Stem Cell Center, Department of Experimental Medical Science, Faculty of Medicine, Lund University, Lund, Sweden
| | - Alessandro Fiorenzano
- Developmental and Regenerative Neurobiology, Lund Stem Cell Center, Department of Experimental Medical Science, Faculty of Medicine, Lund University, Lund, Sweden
| | - Janko Kajtez
- Developmental and Regenerative Neurobiology, Lund Stem Cell Center, Department of Experimental Medical Science, Faculty of Medicine, Lund University, Lund, Sweden
| | - Marcus Davidsson
- Molecular Neuromodulation, Department of Experimental Medical Science, Faculty of Medicine, Lund University, Lund, Sweden
| | - Malin Parmar
- Developmental and Regenerative Neurobiology, Lund Stem Cell Center, Department of Experimental Medical Science, Faculty of Medicine, Lund University, Lund, Sweden
| | - Tomas Björklund
- Molecular Neuromodulation, Department of Experimental Medical Science, Faculty of Medicine, Lund University, Lund, Sweden
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Thanaskody K, Natashah FN, Nordin F, Kamarul Zaman WSW, Tye GJ. Designing molecules: directing stem cell differentiation. Front Bioeng Biotechnol 2024; 12:1396405. [PMID: 38803845 PMCID: PMC11129639 DOI: 10.3389/fbioe.2024.1396405] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2024] [Accepted: 04/23/2024] [Indexed: 05/29/2024] Open
Abstract
Stem cells have been widely applied in regenerative and therapeutic medicine for their unique regenerative properties. Although much research has shown their potential, it remains tricky in directing stem cell differentiation. The advancement of genetic and therapeutic technologies, however, has facilitated this issue through development of design molecules. These molecules are designed to overcome the drawbacks previously faced, such as unexpected differentiation outcomes and insufficient migration of endogenous or exogenous MSCs. Here, we introduced aptamer, bacteriophage, and biological vectors as design molecules and described their characteristics. The methods of designing/developing discussed include various Systematic Evolution of Ligands by Exponential Enrichment (SELEX) procedures, in silico approaches, and non-SELEX methods for aptamers, and genetic engineering methods such as homologous recombination, Bacteriophage Recombineering of Electroporated DNA (BRED), Bacteriophage Recombineering with Infectious Particles (BRIP), and genome rebooting for bacteriophage. For biological vectors, methods such as alternate splicing, multiple promoters, internal ribosomal entry site, CRISPR-Cas9 system and Cre recombinase mediated recombination were used to design viral vectors, while non-viral vectors like exosomes are generated through parental cell-based direct engineering. Besides that, we also discussed the pros and cons, and applications of each design molecule in directing stem cell differentiation to illustrate their great potential in stem cells research. Finally, we highlighted some safety and efficacy concerns to be considered for future studies.
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Affiliation(s)
- Kalaiselvaan Thanaskody
- Centre for Tissue Engineering and Regenerative Medicine (CTERM), Faculty of Medicine, National University of Malaysia, Kuala Lumpur, Malaysia
| | - Fajriyah Nur Natashah
- Centre for Tissue Engineering and Regenerative Medicine (CTERM), Faculty of Medicine, National University of Malaysia, Kuala Lumpur, Malaysia
| | - Fazlina Nordin
- Centre for Tissue Engineering and Regenerative Medicine (CTERM), Faculty of Medicine, National University of Malaysia, Kuala Lumpur, Malaysia
| | - Wan Safwani Wan Kamarul Zaman
- Department of Pharmaceutical Life Sciences, Faculty of Pharmacy, Universiti Malaya, Kuala Lumpur, Malaysia
- Centre for Innovation in Medical Engineering (CIME), Department of Biomedical Engineering, Faculty of Engineering, Universiti Malaya, Kuala Lumpur, Malaysia
| | - Gee Jun Tye
- Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Gelugor, Malaysia
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Da A, Chu Y, Krach J, Liu Y, Park Y, Lee SE. Optical Penetration of Shape-Controlled Metallic Nanosensors across Membrane Barriers. SENSORS (BASEL, SWITZERLAND) 2023; 23:2824. [PMID: 36905027 PMCID: PMC10007193 DOI: 10.3390/s23052824] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 02/06/2023] [Revised: 02/24/2023] [Accepted: 03/01/2023] [Indexed: 06/18/2023]
Abstract
Precise nanostructure geometry that enables the optical biomolecular delivery of nanosensors to the living intracellular environment is highly desirable for precision biological and clinical therapies. However, the optical delivery through membrane barriers utilizing nanosensors remains difficult due to a lack of design guidelines to avoid inherent conflict between optical force and photothermal heat generation in metallic nanosensors during the process. Here, we present a numerical study reporting significantly enhanced optical penetration of nanosensors by engineering nanostructure geometry with minimized photothermal heating generation for penetrating across membrane barriers. We show that by varying the nanosensor geometry, penetration depths can be maximized while heat generated during the penetration process can be minimized. We demonstrate the effect of lateral stress induced by an angularly rotating nanosensor on a membrane barrier by theoretical analysis. Furthermore, we show that by varying the nanosensor geometry, maximized local stress fields at the nanoparticle-membrane interface enhanced the optical penetration process by four-fold. Owing to the high efficiency and stability, we anticipate that precise optical penetration of nanosensors to specific intracellular locations will be beneficial for biological and therapeutic applications.
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Affiliation(s)
- Ancheng Da
- Department of Electrical & Computer Engineering, Biomedical Engineering, Biointerfaces Institute, Applied Physics, Macromolecular Science & Engineering, University of Michigan, Ann Arbor, MI 48109, USA
| | - Yanan Chu
- Department of Electrical & Computer Engineering, Biomedical Engineering, Biointerfaces Institute, Applied Physics, Macromolecular Science & Engineering, University of Michigan, Ann Arbor, MI 48109, USA
| | - Jacob Krach
- Department of Electrical & Computer Engineering, Biomedical Engineering, Biointerfaces Institute, Applied Physics, Macromolecular Science & Engineering, University of Michigan, Ann Arbor, MI 48109, USA
| | - Yunbo Liu
- Department of Electrical & Computer Engineering, Biomedical Engineering, Biointerfaces Institute, Applied Physics, Macromolecular Science & Engineering, University of Michigan, Ann Arbor, MI 48109, USA
| | - Younggeun Park
- Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
| | - Somin Eunice Lee
- Department of Electrical & Computer Engineering, Biomedical Engineering, Biointerfaces Institute, Applied Physics, Macromolecular Science & Engineering, University of Michigan, Ann Arbor, MI 48109, USA
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Ertl HCJ. Mitigating Serious Adverse Events in Gene Therapy with AAV Vectors: Vector Dose and Immunosuppression. Drugs 2023; 83:287-298. [PMID: 36715794 DOI: 10.1007/s40265-023-01836-1] [Citation(s) in RCA: 17] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 01/12/2023] [Indexed: 01/31/2023]
Abstract
Gene transfer with high doses of adeno-associated viral (AAV) vectors has resulted in serious adverse events and even death of the recipients. Toxicity could most likely be circumvented by repeated injections of lower and less toxic doses of vectors. This has not been pursued as AAV vectors induce potent neutralizing antibodies, which prevent cell transduction upon reinjection of the same vector. This review discusses different types of immune responses against AAV vectors and how they offer targets for the elimination or inhibition of vector-specific neutralizing antibodies. Such antibodies can be circumvented by using different virus serotypes for sequential injections, they can be removed by plasmapheresis, or they can be destroyed by enzymatic degradation. Antibody producing cells can be eliminated by proteasome inhibitors. Drugs that inhibit T-cell responses, B-cell signaling, or presentation of the vector's antigens to B cells can prevent or reduce induction of AAV-specific antibodies. Combinations of different approaches and drugs are likely needed to suppress or eliminate neutralizing antibodies, which would then allow for repeated dosing. Alternatively, novel AAV vectors with higher transduction efficacy are being developed and may allow for a dose reduction, although it remains unknown if this will completely address the problem of high-dose adverse events.
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Calvo-López T, Grueso E, Sánchez-Martínez C, Almendral JM. Intracellular virion traffic to the endosome driven by cell type specific sialic acid receptors determines parvovirus tropism. Front Microbiol 2023; 13:1063706. [PMID: 36756201 PMCID: PMC9899843 DOI: 10.3389/fmicb.2022.1063706] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2022] [Accepted: 12/28/2022] [Indexed: 01/24/2023] Open
Abstract
Parvoviruses are promising anticancer and gene therapy agents, but a deep knowledge of the entry process is crucial to exploit their therapeutic potential. We addressed this issue while attempting to retarget the oncolytic parvovirus minute virus of mice (MVMp) to the tumor vasculature. Residues at three functional domains of the icosahedral capsid were substituted by rational design with peptides competing with the vascular endothelial growth factor. Most substitutions impaired virus maturation, though some yielded infectious chimeric virions, and substitutions in a dimple at the twofold axis that allocates sialic acid (SIA) receptors altered viral tropism. One dimple-modified chimeric virion was efficiently attached as MVMp to α2-linked SIA moieties, but the infection was impaired by the binding to some inhibitory α2-3,-6,-8 SIA pseudoreceptors, which hampers intracellular virus traffic to the endosome in a cell type-dependent manner. Infectious from nonproductive traffic could be mechanistically discriminated by an endosomal drastic capsid structural transition comprising the cleavage of some VP2-Nt sequences and its associated VP1-Nt exposure. Correspondingly, neuraminidase removal of inhibitory SIA moieties enhanced the infection quantitatively, correlating to the restored virus traffic to the endosome and the extent of VP2-Nt cleavage/VP1-Nt exposure. This study illustrates (i) structural constraints to retarget parvoviruses with evolutionary adopted narrow grooves allocating small SIA receptors, (ii) the possibility to enhance parvovirus oncolysis by relaxing the glycan network on the cancer cell surface, and (iii) the major role played by the attachment to cell type-specific SIAs in the intracellular virus traffic to the endosome, which may determine parvovirus tropism and host range.
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Affiliation(s)
- Tania Calvo-López
- Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain,Departamento de Biología Molecular, Universidad Autónoma de Madrid, Madrid, Spain
| | - Esther Grueso
- Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain,Departamento de Biología Molecular, Universidad Autónoma de Madrid, Madrid, Spain
| | - Cristina Sánchez-Martínez
- Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain,Departamento de Biología Molecular, Universidad Autónoma de Madrid, Madrid, Spain
| | - José M. Almendral
- Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain,Departamento de Biología Molecular, Universidad Autónoma de Madrid, Madrid, Spain,*Correspondence: José M. Almendral ✉
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8
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Zou L, Wang J, Fang Y, Tian H. PEG-mediated transduction of rAAV as a platform for spatially confined and efficient gene delivery. Biomater Res 2022; 26:69. [PMID: 36461117 PMCID: PMC9716683 DOI: 10.1186/s40824-022-00322-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2022] [Accepted: 11/13/2022] [Indexed: 12/04/2022] Open
Abstract
BACKGROUND Recombinant adeno-associated viruses (rAAV) are commonly used vectors for gene delivery in both basic neuroscience and clinical applications due to their nonpathogenic, minimally immunogenic, and sustained expression properties. However, several challenges remain for the wide-scale rAAV applications, including poor infection of many clinically important cell lines, insufficient expression at low titers, and diffusive transduction in vivo. METHODS In this work, PEG, which is a safe and non-toxic polymer of ethylene oxide monomer, was applied as an auxiliary transduction agent to improve the expression of rAAV. In detail, a small dose of PEG was added into the rAAV solution for the transgene expression in cell lines in vitro, and in the central nervous system (CNS) in vivo. The biocompatibility of PEG enhancer was assessed by characterizing the immune responses, cell morphology, cell tropism of rAAV, neuronal apoptosis, as well as motor function of animals. RESULTS The results show that small dose of PEG additive can effectively improve the gene expression characteristics of rAAV both in vitro and in vivo. Specifically, the PEG additive allows efficient transgene expression in cell lines that are difficult to be transfected with rAAV alone. In vivo studies show that the PEG additive can promote a spatially confined and efficient transgene expression of low-titer rAAV in the brain over long terms. In addition, no obvious side effects of PEG were observed on CNS in the biocompatibility studies. CONCLUSIONS This spatially confined and efficient transduction method can facilitate the applications of rAAV in fundamental research, especially in the precise dissection of neural circuits, and also improve the capabilities of rAAV in the treatment of neurological diseases which originate from the disorders of small nuclei in the brain.
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Affiliation(s)
- Liang Zou
- grid.419265.d0000 0004 1806 6075CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing, 100190 China ,grid.9227.e0000000119573309CAS Center for Excellence in Brain Science and Intelligence Technology, Institute of Neuroscience, Chinese Academy of Sciences, Shanghai, 200031 China ,grid.410726.60000 0004 1797 8419University of Chinese Academy of Sciences, Beijing, 100049 China
| | - Jinfen Wang
- grid.419265.d0000 0004 1806 6075CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing, 100190 China
| | - Ying Fang
- grid.419265.d0000 0004 1806 6075CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing, 100190 China ,grid.9227.e0000000119573309CAS Center for Excellence in Brain Science and Intelligence Technology, Institute of Neuroscience, Chinese Academy of Sciences, Shanghai, 200031 China ,grid.410726.60000 0004 1797 8419University of Chinese Academy of Sciences, Beijing, 100049 China
| | - Huihui Tian
- grid.419265.d0000 0004 1806 6075CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing, 100190 China
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Flitsch LJ, Börner K, Stüllein C, Ziegler S, Sonntag-Buck V, Wiedtke E, Semkova V, Au Yeung SWC, Schlee J, Hajo M, Mathews M, Ludwig BS, Kossatz S, Kessler H, Grimm D, Brüstle O. Identification of adeno-associated virus variants for gene transfer into human neural cell types by parallel capsid screening. Sci Rep 2022; 12:8356. [PMID: 35589936 PMCID: PMC9120183 DOI: 10.1038/s41598-022-12404-0] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2021] [Accepted: 05/09/2022] [Indexed: 12/11/2022] Open
Abstract
Human brain cells generated by in vitro cell programming provide exciting prospects for disease modeling, drug discovery and cell therapy. These applications frequently require efficient and clinically compliant tools for genetic modification of the cells. Recombinant adeno-associated viruses (AAVs) fulfill these prerequisites for a number of reasons, including the availability of a myriad of AAV capsid variants with distinct cell type specificity (also called tropism). Here, we harnessed a customizable parallel screening approach to assess a panel of natural or synthetic AAV capsid variants for their efficacy in lineage-related human neural cell types. We identified common lead candidates suited for the transduction of directly converted, early-stage induced neural stem cells (iNSCs), induced pluripotent stem cell (iPSC)-derived later-stage, radial glia-like neural progenitors, as well as differentiated astrocytic and mixed neuroglial cultures. We then selected a subset of these candidates for functional validation in iNSCs and iPSC-derived astrocytes, using shRNA-induced downregulation of the citrate transporter SLC25A1 and overexpression of the transcription factor NGN2 for proofs-of-concept. Our study provides a comparative overview of the susceptibility of different human cell programming-derived brain cell types to AAV transduction and a critical discussion of the assets and limitations of this specific AAV capsid screening approach.
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Affiliation(s)
- Lea Jessica Flitsch
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty and University Hospital Bonn, Venusberg-Campus 1, Building 76, 53127, Bonn, Germany
| | - Kathleen Börner
- Center for Infectious Diseases, Virology, Medical Faculty, Heidelberg University, Im Neuenheimer Feld 344, 69120, Heidelberg, Germany.,BioQuant, Heidelberg University, Im Neuenheimer Feld 267, 69120, Heidelberg, Germany.,German Center for Infection Research (DZIF), partner site Heidelberg, 69120, Heidelberg, Germany.,AskBio GmbH, Am Taubenfeld 21, 69123, Heidelberg, Germany
| | - Christian Stüllein
- CLADIAC GmbH, Kurfürsten-Anlage 52-58, 69115, Heidelberg, Germany.,Stüllein Software Engineering (SSE), Friedrich-Hartung-Str. 16, 64560, Riedstadt, Germany
| | - Simon Ziegler
- CLADIAC GmbH, Kurfürsten-Anlage 52-58, 69115, Heidelberg, Germany.,KINSYS GmbH, Holtzstr. 2, 76135, Karlsruhe, Germany
| | - Vera Sonntag-Buck
- Center for Infectious Diseases, Virology, Medical Faculty, Heidelberg University, Im Neuenheimer Feld 344, 69120, Heidelberg, Germany.,BioQuant, Heidelberg University, Im Neuenheimer Feld 267, 69120, Heidelberg, Germany.,German Center for Infection Research (DZIF), partner site Heidelberg, 69120, Heidelberg, Germany
| | - Ellen Wiedtke
- Center for Infectious Diseases, Virology, Medical Faculty, Heidelberg University, Im Neuenheimer Feld 344, 69120, Heidelberg, Germany.,BioQuant, Heidelberg University, Im Neuenheimer Feld 267, 69120, Heidelberg, Germany
| | - Vesselina Semkova
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty and University Hospital Bonn, Venusberg-Campus 1, Building 76, 53127, Bonn, Germany.,LIFE and BRAIN GmbH, Venusberg-Campus 1, Building 76, 53127, Bonn, Germany
| | - Si Wah Christina Au Yeung
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty and University Hospital Bonn, Venusberg-Campus 1, Building 76, 53127, Bonn, Germany
| | - Julia Schlee
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty and University Hospital Bonn, Venusberg-Campus 1, Building 76, 53127, Bonn, Germany
| | - Mohamad Hajo
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty and University Hospital Bonn, Venusberg-Campus 1, Building 76, 53127, Bonn, Germany.,Federal Institute for Drugs and Medical Devices (BfArM), Kurt-Georg-Kiesinger-Allee 3, 53175, Bonn, Germany
| | - Mona Mathews
- LIFE and BRAIN GmbH, Venusberg-Campus 1, Building 76, 53127, Bonn, Germany
| | - Beatrice Stefanie Ludwig
- Department of Nuclear Medicine, School of Medicine, Technical University Munich (TUM), University Hospital Klinikum Rechts der Isar and Central Institute for Translational Cancer Research (Transla TUM, Einsteinstr. 25, 81675, Munich, Germany
| | - Susanne Kossatz
- Department of Nuclear Medicine, School of Medicine, Technical University Munich (TUM), University Hospital Klinikum Rechts der Isar and Central Institute for Translational Cancer Research (Transla TUM, Einsteinstr. 25, 81675, Munich, Germany
| | - Horst Kessler
- Institute for Advanced Study, Department Chemie, Technical University Munich (TUM), Lichtenbergstr. 4, 85747, Garching, Germany
| | - Dirk Grimm
- Center for Infectious Diseases, Virology, Medical Faculty, Heidelberg University, Im Neuenheimer Feld 344, 69120, Heidelberg, Germany. .,BioQuant, Heidelberg University, Im Neuenheimer Feld 267, 69120, Heidelberg, Germany. .,German Center for Infection Research (DZIF), partner site Heidelberg, 69120, Heidelberg, Germany. .,German Center for Cardiovascular Research (DZHK), partner site Heidelberg, 69120, Heidelberg, Germany.
| | - Oliver Brüstle
- Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty and University Hospital Bonn, Venusberg-Campus 1, Building 76, 53127, Bonn, Germany. .,LIFE and BRAIN GmbH, Venusberg-Campus 1, Building 76, 53127, Bonn, Germany.
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10
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Singh V, Khan N, Jayandharan GR. Vector engineering, strategies and targets in cancer gene therapy. Cancer Gene Ther 2022; 29:402-417. [PMID: 33859378 DOI: 10.1038/s41417-021-00331-7] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2020] [Revised: 02/23/2021] [Accepted: 03/24/2021] [Indexed: 02/02/2023]
Abstract
Understanding the molecular basis of disease and the design of rationally designed molecular therapies has been the holy grail in the management of human cancers. Gene-based therapies are an important avenue for achieving a possible cure. Focused research in the last three decades has provided significant clues to optimize the potential of cancer gene therapy. The development of gene therapies with a high potential to kill the target cells at the lowest effective dose possible, the development of vectors with significant ability to target cancer-associated antigen, the application of adjunct therapies to target dysregulated microRNA, and embracing a hybrid strategy with a combination of gene therapy and low-dose chemotherapy in a disease-specific manner will be pivotal. This article outlines the advances and challenges in the field with emphasis on the biology and scope of vectors used for gene transfer, newer targets identified, and their outcome in preclinical and clinical studies.
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Affiliation(s)
- Vijayata Singh
- Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, UP, India
| | - Nusrat Khan
- Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, UP, India
| | - Giridhara R Jayandharan
- Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, UP, India. .,The Mehta Family Centre for Engineering in Medicine, Indian Institute of Technology, Kanpur, UP, India.
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11
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Mnyandu N, Limani SW, Arbuthnot P, Maepa MB. Advances in designing Adeno-associated viral vectors for development of anti-HBV gene therapeutics. Virol J 2021; 18:247. [PMID: 34903258 PMCID: PMC8670254 DOI: 10.1186/s12985-021-01715-9] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2021] [Accepted: 11/26/2021] [Indexed: 12/25/2022] Open
Abstract
Despite the five decades having passed since discovery of the hepatitis B virus (HBV), together with development of an effective anti-HBV vaccine, infection with the virus remains a serious public health problem and results in nearly 900,000 annual deaths worldwide. Current therapies do not eliminate the virus and viral replication typically reactivates after treatment withdrawal. Hence, current endeavours are aimed at developing novel therapies to achieve a functional cure. Nucleic acid-based therapeutic approaches are promising, with several candidates showing excellent potencies in preclinical and early stages of clinical development. However, this class of therapeutics is yet to become part of standard anti-HBV treatment regimens. Obstacles delaying development of gene-based therapies include lack of clinically relevant delivery methods and a paucity of good animal models for preclinical characterisation. Recent studies have demonstrated safety and efficiency of Adeno-associated viral vectors (AAVs) in gene therapy. However, AAVs do have flaws and this has prompted research aimed at improving design of novel and artificially synthesised AAVs. Main goals are to improve liver transduction efficiencies and avoiding immune clearance. Application of AAVs to model HBV replication in vivo is also useful for characterising anti-HBV gene therapeutics. This review summarises recent advances in AAV engineering and their contributions to progress with anti-HBV gene therapy development.
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Affiliation(s)
- Njabulo Mnyandu
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
| | - Shonisani Wendy Limani
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
| | - Patrick Arbuthnot
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
| | - Mohube Betty Maepa
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa.
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12
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Wagner HJ, Weber W, Fussenegger M. Synthetic Biology: Emerging Concepts to Design and Advance Adeno-Associated Viral Vectors for Gene Therapy. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2021; 8:2004018. [PMID: 33977059 PMCID: PMC8097373 DOI: 10.1002/advs.202004018] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/20/2020] [Revised: 12/18/2020] [Indexed: 05/28/2023]
Abstract
Three recent approvals and over 100 ongoing clinical trials make adeno-associated virus (AAV)-based vectors the leading gene delivery vehicles in gene therapy. Pharmaceutical companies are investing in this small and nonpathogenic gene shuttle to increase the therapeutic portfolios within the coming years. This prospect of marking a new era in gene therapy has fostered both investigations of the fundamental AAV biology as well as engineering studies to enhance delivery vehicles. Driven by the high clinical potential, a new generation of synthetic-biologically engineered AAV vectors is on the rise. Concepts from synthetic biology enable the control and fine-tuning of vector function at different stages of cellular transduction and gene expression. It is anticipated that the emerging field of synthetic-biologically engineered AAV vectors can shape future gene therapeutic approaches and thus the design of tomorrow's gene delivery vectors. This review describes and discusses the recent trends in capsid and vector genome engineering, with particular emphasis on synthetic-biological approaches.
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Affiliation(s)
- Hanna J. Wagner
- Department of Biosystems Science and EngineeringETH ZurichMattenstrasse 26Basel4058Switzerland
- Faculty of BiologyUniversity of FreiburgSchänzlestraße 1Freiburg79104Germany
- Signalling Research Centres BIOSS and CIBSSUniversity of FreiburgSchänzlestraße 18Freiburg79104Germany
| | - Wilfried Weber
- Faculty of BiologyUniversity of FreiburgSchänzlestraße 1Freiburg79104Germany
- Signalling Research Centres BIOSS and CIBSSUniversity of FreiburgSchänzlestraße 18Freiburg79104Germany
| | - Martin Fussenegger
- Department of Biosystems Science and EngineeringETH ZurichMattenstrasse 26Basel4058Switzerland
- Faculty of ScienceUniversity of BaselKlingelbergstrasse 50Basel4056Switzerland
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13
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Zhu D, Schieferecke AJ, Lopez PA, Schaffer DV. Adeno-Associated Virus Vector for Central Nervous System Gene Therapy. Trends Mol Med 2021; 27:524-537. [PMID: 33895085 DOI: 10.1016/j.molmed.2021.03.010] [Citation(s) in RCA: 42] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2020] [Revised: 03/15/2021] [Accepted: 03/22/2021] [Indexed: 12/14/2022]
Abstract
The past several years have witnessed significant advances in the development of therapeutic gene delivery for neurological disorders of the central nervous system (CNS). In particular, genome-wide sequencing analysis has deepened our understanding of mutations that underlie many monogenic disorders, which in turn has contributed to clinical advances involving adeno-associated virus (AAV) vector delivery of replacement genes to treat recessive disorders. Moreover, gene therapy has been further bolstered with advances in genome editing tools that allow researchers to silence, repair, and amend endogenous genes. However, despite strong preclinical and clinical progress, challenges remain, including delivery and safety. Here, we discuss advances in AAV engineering, recent developments in cargo design, and translation of these technologies towards clinical progress.
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Affiliation(s)
- Danqing Zhu
- California Institute for Quantitative Biosciences, University of California, Berkeley, CA, 94720, USA
| | - Adam J Schieferecke
- Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720, USA
| | - Paola A Lopez
- Department of Bioengineering, University of California, Berkeley, CA, 94720, USA
| | - David V Schaffer
- California Institute for Quantitative Biosciences, University of California, Berkeley, CA, 94720, USA; Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720, USA; Department of Bioengineering, University of California, Berkeley, CA, 94720, USA; Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, 94720, USA; Helen Wills Neuroscience Institute, University of California, Berkeley, CA, 94720, USA.
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14
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Cappella M, Elouej S, Biferi MG. The Potential of Induced Pluripotent Stem Cells to Test Gene Therapy Approaches for Neuromuscular and Motor Neuron Disorders. Front Cell Dev Biol 2021; 9:662837. [PMID: 33937264 PMCID: PMC8080375 DOI: 10.3389/fcell.2021.662837] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2021] [Accepted: 03/22/2021] [Indexed: 12/11/2022] Open
Abstract
The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) represents a major advance for the development of human disease models. The emerging of this technique fostered the concept of "disease in a dish," which consists into the generation of patient-specific models in vitro. Currently, iPSCs are used to study pathological molecular mechanisms caused by genetic mutations and they are considered a reliable model for high-throughput drug screenings. Importantly, precision-medicine approaches to treat monogenic disorders exploit iPSCs potential for the selection and validation of lead candidates. For example, antisense oligonucleotides (ASOs) were tested with promising results in myoblasts or motor neurons differentiated from iPSCs of patients affected by either Duchenne muscular dystrophy or Amyotrophic lateral sclerosis. However, the use of iPSCs needs additional optimization to ensure translational success of the innovative strategies based on gene delivery through adeno associated viral vectors (AAV) for these diseases. Indeed, to establish an efficient transduction of iPSCs with AAV, several aspects should be optimized, including viral vector serotype, viral concentration and timing of transduction. This review will outline the use of iPSCs as a model for the development and testing of gene therapies for neuromuscular and motor neuron disorders. It will then discuss the advantages for the use of this versatile tool for gene therapy, along with the challenges associated with the viral vector transduction of iPSCs.
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Affiliation(s)
- Marisa Cappella
- Sorbonne University, INSERM, Institute of Myology, Center of Research in Myology, Paris, France
| | - Sahar Elouej
- Sorbonne University, INSERM, Institute of Myology, Center of Research in Myology, Paris, France
| | - Maria Grazia Biferi
- Sorbonne University, INSERM, Institute of Myology, Center of Research in Myology, Paris, France
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15
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Marques AD, Kummer M, Kondratov O, Banerjee A, Moskalenko O, Zolotukhin S. Applying machine learning to predict viral assembly for adeno-associated virus capsid libraries. Mol Ther Methods Clin Dev 2021; 20:276-286. [PMID: 33511242 PMCID: PMC7809249 DOI: 10.1016/j.omtm.2020.11.017] [Citation(s) in RCA: 33] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2020] [Accepted: 11/24/2020] [Indexed: 12/12/2022]
Abstract
Machine learning (ML) can aid in novel discoveries in the field of viral gene therapy. Specifically, big data gathered through next-generation sequencing (NGS) of complex capsid libraries is an especially prominent source of lost potential in data analysis and prediction. Furthermore, adeno-associated virus (AAV)-based capsid libraries are becoming increasingly popular as a tool to select candidates for gene therapy vectors. These higher complexity AAV capsid libraries have previously been created and selected in vivo; however, in silico analysis using ML computer algorithms may augment smarter and more robust libraries for selection. In this study, data of AAV capsid libraries gathered before and after viral assembly are used to train ML algorithms. We found that two ML computer algorithms, artificial neural networks (ANNs), and support vector machines (SVMs), can be trained to predict whether unknown capsid variants may assemble into viable virus-like structures. Using the most accurate models constructed, hypothetical mutation patterns in library construction were simulated to suggest the importance of N495, G546, and I554 in AAV2-derived capsids. Finally, two comparative libraries were generated using ML-derived data to biologically validate these findings and demonstrate the predictive power of ML in vector design.
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Affiliation(s)
- Andrew D. Marques
- Department of Pediatrics, Division of Cellular and Molecular Therapy, University of Florida, Gainesville, FL 32608, USA
| | - Michael Kummer
- Department of Computer & Information Science & Engineering, University of Florida, Gainesville, FL 32603, USA
| | - Oleksandr Kondratov
- Department of Pediatrics, Division of Cellular and Molecular Therapy, University of Florida, Gainesville, FL 32608, USA
| | - Arunava Banerjee
- Department of Computer & Information Science & Engineering, University of Florida, Gainesville, FL 32603, USA
| | - Oleksandr Moskalenko
- University of Florida Research Computing, University of Florida, Gainesville, FL 32608, USA
| | - Sergei Zolotukhin
- Department of Pediatrics, Division of Cellular and Molecular Therapy, University of Florida, Gainesville, FL 32608, USA
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16
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Chowdhury EA, Meno-Tetang G, Chang HY, Wu S, Huang HW, Jamier T, Chandran J, Shah DK. Current progress and limitations of AAV mediated delivery of protein therapeutic genes and the importance of developing quantitative pharmacokinetic/pharmacodynamic (PK/PD) models. Adv Drug Deliv Rev 2021; 170:214-237. [PMID: 33486008 DOI: 10.1016/j.addr.2021.01.017] [Citation(s) in RCA: 44] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2020] [Revised: 01/13/2021] [Accepted: 01/14/2021] [Indexed: 12/17/2022]
Abstract
While protein therapeutics are one of the most successful class of drug molecules, they are expensive and not suited for treating chronic disorders that require long-term dosing. Adeno-associated virus (AAV) mediated in vivo gene therapy represents a viable alternative, which can deliver the genes of protein therapeutics to produce long-term expression of proteins in target tissues. Ongoing clinical trials and recent regulatory approvals demonstrate great interest in these therapeutics, however, there is a lack of understanding regarding their cellular disposition, whole-body disposition, dose-exposure relationship, exposure-response relationship, and how product quality and immunogenicity affects these important properties. In addition, there is a lack of quantitative studies to support the development of pharmacokinetic-pharmacodynamic models, which can support the discovery, development, and clinical translation of this delivery system. In this review, we have provided a state-of-the-art overview of current progress and limitations related to AAV mediated delivery of protein therapeutic genes, along with our perspective on the steps that need to be taken to improve clinical translation of this therapeutic modality.
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17
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Rodríguez-Márquez E, Meumann N, Büning H. Adeno-associated virus (AAV) capsid engineering in liver-directed gene therapy. Expert Opin Biol Ther 2020; 21:749-766. [PMID: 33331201 DOI: 10.1080/14712598.2021.1865303] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Introduction: Gene therapy clinical trials with adeno-associated virus (AAV) vectors report impressive clinical efficacy data. Nevertheless, challenges have become apparent, such as the need for high vector doses and the induction of anti-AAV immune responses that cause the loss of vector-transduced hepatocytes. This fostered research focusing on development of next-generation AAV vectors capable of dealing with these hurdles.Areas Covered: While both the viral vector genome and the capsid are subjects to engineering, this review focuses on the latter. Specifically, we summarize the principles of capsid engineering strategies, and describe developments and applications of engineered capsid variants for liver-directed gene therapy.Expert Opinion: Capsid engineering is a promising strategy to significantly improve efficacy of the AAV vector system in clinical application. Reduction in vector dose will further improve vector safety, lower the risk of host immune responses and the cost of manufacturing. Capsid engineering is also expected to result in AAV vectors applicable to patients with preexisting immunity toward natural AAV serotypes.
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Affiliation(s)
- Esther Rodríguez-Márquez
- Universidad Autónoma De Madrid, Madrid, Spain.,Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.,REBIRTH Research Center for Translational Regenerative Medicine, Hannover Medical School, Hannover, Germany
| | - Nadja Meumann
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.,REBIRTH Research Center for Translational Regenerative Medicine, Hannover Medical School, Hannover, Germany
| | - Hildegard Büning
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.,REBIRTH Research Center for Translational Regenerative Medicine, Hannover Medical School, Hannover, Germany.,Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.,German Center for Infection Research (DZIF, Partner Site Hannover-Braunschweig, Germany
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18
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Carneiro A, Lee H, Lin L, van Haasteren J, Schaffer DV. Novel Lung Tropic Adeno-Associated Virus Capsids for Therapeutic Gene Delivery. Hum Gene Ther 2020; 31:996-1009. [PMID: 32799685 DOI: 10.1089/hum.2020.169] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Efforts to identify mutations that underlie inherited genetic diseases combined with strides in the development of gene therapy vectors over the last three decades have culminated in the approval of several adeno-associated virus (AAV)-based gene therapies. Genetic diseases that manifest in the lung such as cystic fibrosis (CF) and surfactant deficiencies, however, have so far proven to be elusive targets. Early clinical trials in CF using AAV serotype 2 (AAV2) achieved safety, but not efficacy endpoints; however, importantly, these studies provided critical information on barriers that need to be surmounted to translate AAV lung gene therapy toward clinical success. Bolstered with an improved understanding of AAV biology and more clinically relevant lung models, next-generation molecular biology and bioinformatics approaches have given rise to novel AAV capsid variants that offer improvements in transduction efficiency, immunological profile, and the ability to circumvent physical barriers in the lung such as mucus. This review discusses the principal limiting barriers to clinical success in lung gene therapy and focuses on novel engineered AAV capsid variants that have been developed to overcome those challenges.
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Affiliation(s)
- Ana Carneiro
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, USA
| | - Hyuncheol Lee
- California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, California, USA
| | - Li Lin
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, USA
| | - Joost van Haasteren
- California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, California, USA
| | - David V Schaffer
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, USA.,California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, California, USA.,Department of Bioengineering, University of California, Berkeley, California, USA.,Department of Molecular and Cell Biology, University of California, Berkeley, California, USA.,Helen Wills Neuroscience Institute, University of California, Berkeley, California, USA.,Innovative Genomics Institute (IGI), University of California, Berkeley, California, USA
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19
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Brommel CM, Cooney AL, Sinn PL. Adeno-Associated Virus-Based Gene Therapy for Lifelong Correction of Genetic Disease. Hum Gene Ther 2020; 31:985-995. [PMID: 32718227 PMCID: PMC7495917 DOI: 10.1089/hum.2020.138] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2020] [Accepted: 07/27/2020] [Indexed: 12/27/2022] Open
Abstract
The list of successful gene therapy trials using adeno-associated virus (AAV)-based vectors continues to grow and includes a wide range of monogenic diseases. Replication incompetent AAV genomes typically remain episomal and expression dilutes as cells divide and die. Consequently, long-term transgene expression from AAV is best suited for quiescent cell types, such as retinal cells, myocytes, or neurons. For genetic diseases that involve cells with steady turnover, AAV-conferred correction may require routine readministration, where every dose carries the risk of developing an adaptive immune response that renders treatment ineffective. Here, we discuss innovative approaches to permanently modify the host genome using AAV-based platforms, thus potentially requiring only a single dose. Such approaches include using AAV delivery of DNA transposons, homologous recombination templates into safe harbors, and nucleases for targeting integration. In tissues with continual cell turnover, genetic modification of progenitor cell populations will help ensure persistent therapeutic outcomes. Combining the safety profile of AAV-based gene therapy vectors with the ability to integrate a therapeutic transgene creates novel solutions to the challenge of lifelong curative treatments for human genetic diseases.
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Affiliation(s)
| | - Ashley L. Cooney
- Department of Pediatrics, University of Iowa, Iowa City, Iowa, USA
| | - Patrick L. Sinn
- Program in Molecular Medicine, University of Iowa, Iowa City, Iowa, USA
- Department of Pediatrics, University of Iowa, Iowa City, Iowa, USA
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20
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Li D, Yang H, Xiong F, Xu X, Zeng WB, Zhao F, Luo MH. Anterograde Neuronal Circuit Tracers Derived from Herpes Simplex Virus 1: Development, Application, and Perspectives. Int J Mol Sci 2020; 21:E5937. [PMID: 32824837 PMCID: PMC7460661 DOI: 10.3390/ijms21165937] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2020] [Revised: 08/16/2020] [Accepted: 08/17/2020] [Indexed: 12/19/2022] Open
Abstract
Herpes simplex virus type 1 (HSV-1) has great potential to be applied as a viral tool for gene delivery or oncolysis. The broad infection tropism of HSV-1 makes it a suitable tool for targeting many different cell types, and its 150 kb double-stranded DNA genome provides great capacity for exogenous genes. Moreover, the features of neuron infection and neuron-to-neuron spread also offer special value to neuroscience. HSV-1 strain H129, with its predominant anterograde transneuronal transmission, represents one of the most promising anterograde neuronal circuit tracers to map output neuronal pathways. Decades of development have greatly expanded the H129-derived anterograde tracing toolbox, including polysynaptic and monosynaptic tracers with various fluorescent protein labeling. These tracers have been applied to neuroanatomical studies, and have contributed to revealing multiple important neuronal circuits. However, current H129-derived tracers retain intrinsic drawbacks that limit their broad application, such as yet-to-be improved labeling intensity, potential nonspecific retrograde labeling, and high toxicity. The biological complexity of HSV-1 and its insufficiently characterized virological properties have caused difficulties in its improvement and optimization as a viral tool. In this review, we focus on the current H129-derived viral tracers and highlight strategies in which future technological development can advance its use as a tool.
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Affiliation(s)
- Dong Li
- State Key Laboratory of Virology, CAS Center for Excellence in Brain Science and Intelligence Technology, Center for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; (D.L.); (H.Y.); (F.X.); (W.-B.Z.)
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Hong Yang
- State Key Laboratory of Virology, CAS Center for Excellence in Brain Science and Intelligence Technology, Center for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; (D.L.); (H.Y.); (F.X.); (W.-B.Z.)
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Feng Xiong
- State Key Laboratory of Virology, CAS Center for Excellence in Brain Science and Intelligence Technology, Center for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; (D.L.); (H.Y.); (F.X.); (W.-B.Z.)
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Xiangmin Xu
- Department of Anatomy and Neurobiology, School of Medicine, University of California, Irvine, CA 92697-1275, USA;
| | - Wen-Bo Zeng
- State Key Laboratory of Virology, CAS Center for Excellence in Brain Science and Intelligence Technology, Center for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; (D.L.); (H.Y.); (F.X.); (W.-B.Z.)
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Fei Zhao
- School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China
- Chinese Institute for Brain Research, Beijing 102206, China
| | - Min-Hua Luo
- State Key Laboratory of Virology, CAS Center for Excellence in Brain Science and Intelligence Technology, Center for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; (D.L.); (H.Y.); (F.X.); (W.-B.Z.)
- University of Chinese Academy of Sciences, Beijing 100049, China
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21
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Tay LS, Palmer N, Panwala R, Chew WL, Mali P. Translating CRISPR-Cas Therapeutics: Approaches and Challenges. CRISPR J 2020; 3:253-275. [PMID: 32833535 PMCID: PMC7469700 DOI: 10.1089/crispr.2020.0025] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
CRISPR-Cas clinical trials have begun, offering a first glimpse at how DNA and RNA targeting could enable therapies for many genetic and epigenetic human diseases. The speedy progress of CRISPR-Cas from discovery and adoption to clinical use is built on decades of traditional gene therapy research and belies the multiple challenges that could derail the successful translation of these new modalities. Here, we review how CRISPR-Cas therapeutics are translated from technological systems to therapeutic modalities, paying particular attention to the therapeutic cascade from cargo to delivery vector, manufacturing, administration, pipelines, safety, and therapeutic target profiles. We also explore potential solutions to some of the obstacles facing successful CRISPR-Cas translation. We hope to illuminate how CRISPR-Cas is brought from the academic bench toward use in the clinic.
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Affiliation(s)
- Lavina Sierra Tay
- Laboratory of Synthetic Biology and Genome Editing Therapeutics, Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, Singapore
| | - Nathan Palmer
- Division of Biological Sciences, University of California San Diego, La Jolla, California, USA
| | - Rebecca Panwala
- Department of Bioengineering, University of California San Diego, La Jolla, California, USA
| | - Wei Leong Chew
- Laboratory of Synthetic Biology and Genome Editing Therapeutics, Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, Singapore
| | - Prashant Mali
- Department of Bioengineering, University of California San Diego, La Jolla, California, USA
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22
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Martin RM, Ikeda K, Cromer MK, Uchida N, Nishimura T, Romano R, Tong AJ, Lemgart VT, Camarena J, Pavel-Dinu M, Sindhu C, Wiebking V, Vaidyanathan S, Dever DP, Bak RO, Laustsen A, Lesch BJ, Jakobsen MR, Sebastiano V, Nakauchi H, Porteus MH. Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination. Cell Stem Cell 2020; 24:821-828.e5. [PMID: 31051134 DOI: 10.1016/j.stem.2019.04.001] [Citation(s) in RCA: 141] [Impact Index Per Article: 28.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2018] [Revised: 10/08/2018] [Accepted: 03/29/2019] [Indexed: 11/25/2022]
Abstract
Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for in vitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.
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Affiliation(s)
- Renata M Martin
- Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
| | - Kazuya Ikeda
- Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
| | - M Kyle Cromer
- Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
| | - Nobuko Uchida
- ReGen Med Division, BOCO Silicon Valley, Palo Alto, CA 94303, USA
| | | | - Rosa Romano
- Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
| | - Andrew J Tong
- Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
| | - Viktor T Lemgart
- Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
| | - Joab Camarena
- Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
| | - Mara Pavel-Dinu
- Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
| | - Camille Sindhu
- Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
| | - Volker Wiebking
- Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
| | | | - Daniel P Dever
- Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
| | - Rasmus O Bak
- Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
| | - Anders Laustsen
- Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, 8000 Aarhus C, Denmark; Aarhus Research Centre of Innate Immunology, Aarhus University, Wilhelm Meyers Alle 4, 8000 Aarhus C, Denmark
| | - Benjamin J Lesch
- Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
| | - Martin R Jakobsen
- Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, 8000 Aarhus C, Denmark; Aarhus Research Centre of Innate Immunology, Aarhus University, Wilhelm Meyers Alle 4, 8000 Aarhus C, Denmark
| | - Vittorio Sebastiano
- Department of Obstetrics & Gynecology, Stanford University, Stanford, CA 94305, USA
| | | | - Matthew H Porteus
- Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
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23
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Collins LR, Shepard KA. CIRM tools and technologies: Breaking bottlenecks to the development of stem cell therapies. Stem Cells Transl Med 2020; 9:1129-1136. [PMID: 32619326 PMCID: PMC7519770 DOI: 10.1002/sctm.20-0055] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2020] [Revised: 05/06/2020] [Accepted: 05/30/2020] [Indexed: 01/01/2023] Open
Abstract
The California Institute for Regenerative Medicine (CIRM) has a mission to accelerate stem cell treatments to patients with unmet medical needs. This perspective describes successful examples of work funded by CIRM's New Cell Lines and Tools and Technologies Initiatives, which were developed to address bottlenecks to stem cell research and translation. The tools developed through these programs evolved from more discovery-oriented technologies, such as disease models, differentiation processes, and assays, to more translation focused tools, including scalable good manufacturing processes, animal models, and tools for clinical cell delivery. These tools are available to the research community and many are facilitating translation of regenerative therapeutics today.
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Affiliation(s)
- Lila R. Collins
- California Institute for Regenerative MedicineOaklandCaliforniaUSA
| | - Kelly A. Shepard
- California Institute for Regenerative MedicineOaklandCaliforniaUSA
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24
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Li H, Yang Y, Hong W, Huang M, Wu M, Zhao X. Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects. Signal Transduct Target Ther 2020; 5:1. [PMID: 32296011 PMCID: PMC6946647 DOI: 10.1038/s41392-019-0089-y] [Citation(s) in RCA: 1062] [Impact Index Per Article: 212.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2019] [Revised: 09/21/2019] [Accepted: 09/21/2019] [Indexed: 02/06/2023] Open
Abstract
Based on engineered or bacterial nucleases, the development of genome editing technologies has opened up the possibility of directly targeting and modifying genomic sequences in almost all eukaryotic cells. Genome editing has extended our ability to elucidate the contribution of genetics to disease by promoting the creation of more accurate cellular and animal models of pathological processes and has begun to show extraordinary potential in a variety of fields, ranging from basic research to applied biotechnology and biomedical research. Recent progress in developing programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-associated nucleases, has greatly expedited the progress of gene editing from concept to clinical practice. Here, we review recent advances of the three major genome editing technologies (ZFNs, TALENs, and CRISPR/Cas9) and discuss the applications of their derivative reagents as gene editing tools in various human diseases and potential future therapies, focusing on eukaryotic cells and animal models. Finally, we provide an overview of the clinical trials applying genome editing platforms for disease treatment and some of the challenges in the implementation of this technology.
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Affiliation(s)
- Hongyi Li
- Department of Gynecology and Obstetrics, Development and Related Disease of Women and Children Key Laboratory of Sichuan Province, Key Laboratory of Birth Defects and Related Diseases of Women and Children, Ministry of Education, West China Second Hospital, Sichuan University, Chengdu, 610041, P. R. China
| | - Yang Yang
- Department of Gynecology and Obstetrics, Development and Related Disease of Women and Children Key Laboratory of Sichuan Province, Key Laboratory of Birth Defects and Related Diseases of Women and Children, Ministry of Education, West China Second Hospital, Sichuan University, Chengdu, 610041, P. R. China
| | - Weiqi Hong
- Laboratory of Aging Research and Cancer Drug Target, State Key Laboratory of Biotherapy, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, No. 17, Block 3, Southern Renmin Road, Chengdu, Sichuan, 610041, P. R. China
| | - Mengyuan Huang
- Laboratory of Aging Research and Cancer Drug Target, State Key Laboratory of Biotherapy, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, No. 17, Block 3, Southern Renmin Road, Chengdu, Sichuan, 610041, P. R. China
| | - Min Wu
- Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND, 58203, USA.
| | - Xia Zhao
- Department of Gynecology and Obstetrics, Development and Related Disease of Women and Children Key Laboratory of Sichuan Province, Key Laboratory of Birth Defects and Related Diseases of Women and Children, Ministry of Education, West China Second Hospital, Sichuan University, Chengdu, 610041, P. R. China.
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25
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Davidsson M, Wang G, Aldrin-Kirk P, Cardoso T, Nolbrant S, Hartnor M, Mudannayake J, Parmar M, Björklund T. A systematic capsid evolution approach performed in vivo for the design of AAV vectors with tailored properties and tropism. Proc Natl Acad Sci U S A 2019; 116:27053-27062. [PMID: 31818949 PMCID: PMC6936499 DOI: 10.1073/pnas.1910061116] [Citation(s) in RCA: 100] [Impact Index Per Article: 16.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Adeno-associated virus (AAV) capsid modification enables the generation of recombinant vectors with tailored properties and tropism. Most approaches to date depend on random screening, enrichment, and serendipity. The approach explored here, called BRAVE (barcoded rational AAV vector evolution), enables efficient selection of engineered capsid structures on a large scale using only a single screening round in vivo. The approach stands in contrast to previous methods that require multiple generations of enrichment. With the BRAVE approach, each virus particle displays a peptide, derived from a protein, of known function on the AAV capsid surface, and a unique molecular barcode in the packaged genome. The sequencing of RNA-expressed barcodes from a single-generation in vivo screen allows the mapping of putative binding sequences from hundreds of proteins simultaneously. Using the BRAVE approach and hidden Markov model-based clustering, we present 25 synthetic capsid variants with refined properties, such as retrograde axonal transport in specific subtypes of neurons, as shown for both rodent and human dopaminergic neurons.
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Affiliation(s)
- Marcus Davidsson
- Molecular Neuromodulation, Department of Experimental Medical Science, Lund University, 221 84 Lund, Sweden
| | - Gang Wang
- Molecular Neuromodulation, Department of Experimental Medical Science, Lund University, 221 84 Lund, Sweden
| | - Patrick Aldrin-Kirk
- Molecular Neuromodulation, Department of Experimental Medical Science, Lund University, 221 84 Lund, Sweden
| | - Tiago Cardoso
- Developmental and Regenerative Neurobiology, Department of Experimental Medical Science, Lund Stem Cell Center, Lund University, 221 84 Lund, Sweden
| | - Sara Nolbrant
- Developmental and Regenerative Neurobiology, Department of Experimental Medical Science, Lund Stem Cell Center, Lund University, 221 84 Lund, Sweden
| | - Morgan Hartnor
- Molecular Neuromodulation, Department of Experimental Medical Science, Lund University, 221 84 Lund, Sweden
| | - Janitha Mudannayake
- Molecular Neuromodulation, Department of Experimental Medical Science, Lund University, 221 84 Lund, Sweden
| | - Malin Parmar
- Developmental and Regenerative Neurobiology, Department of Experimental Medical Science, Lund Stem Cell Center, Lund University, 221 84 Lund, Sweden
| | - Tomas Björklund
- Molecular Neuromodulation, Department of Experimental Medical Science, Lund University, 221 84 Lund, Sweden
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26
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Kim SH, Lee S, Lee H, Cho M, Schaffer DV, Jang JH. AAVR-Displaying Interfaces: Serotype-Independent Adeno-Associated Virus Capture and Local Delivery Systems. MOLECULAR THERAPY. NUCLEIC ACIDS 2019; 18:432-443. [PMID: 31670142 PMCID: PMC6831863 DOI: 10.1016/j.omtn.2019.09.015] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/28/2018] [Revised: 09/17/2019] [Accepted: 09/17/2019] [Indexed: 12/12/2022]
Abstract
Interfacing gene delivery vehicles with biomaterials has the potential to play a key role in diversifying gene transfer capabilities, including localized, patterned, and controlled delivery. However, strategies for modifying biomaterials to interact with delivery vectors must be redesigned whenever new delivery vehicles and applications are explored. We have developed a vector-independent biomaterial platform capable of interacting with various adeno-associated viral (AAV) serotypes. A water-soluble, cysteine-tagged, recombinant protein version of the recently discovered multi-AAV serotype receptor (AAVR), referred to as cys-AAVR, was conjugated to maleimide-displaying polycaprolactone (PCL) materials using click chemistry. The resulting cys-AAVR-PCL system bound to a broad range of therapeutically relevant AAV serotypes, thereby providing a platform capable of modulating the delivery of all AAV serotypes. Intramuscular injection of cys-AAVR-PCL microspheres with bound AAV vectors resulted in localized and sustained gene delivery as well as reduced spread to off-target organs compared to a vector solution. This cys-AAVR-PCL system is thus an effective approach for biomaterial-based AAV gene delivery for a broad range of therapeutic applications.
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Affiliation(s)
- Seung-Hyun Kim
- Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul 120-749, Korea
| | - Slgirim Lee
- Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul 120-749, Korea; Simpson Querrey Institute for BioNanotechnology, Northwestern University, Chicago, IL 60611, USA
| | - Heehyung Lee
- Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul 120-749, Korea
| | - Mira Cho
- Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul 120-749, Korea
| | - David V Schaffer
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, CA 94720-3220, USA; Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720-3220, USA; Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA 94720-3220, USA.
| | - Jae-Hyung Jang
- Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul 120-749, Korea.
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27
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Pekrun K, De Alencastro G, Luo QJ, Liu J, Kim Y, Nygaard S, Galivo F, Zhang F, Song R, Tiffany MR, Xu J, Hebrok M, Grompe M, Kay MA. Using a barcoded AAV capsid library to select for clinically relevant gene therapy vectors. JCI Insight 2019; 4:131610. [PMID: 31723052 PMCID: PMC6948855 DOI: 10.1172/jci.insight.131610] [Citation(s) in RCA: 68] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2019] [Accepted: 10/08/2019] [Indexed: 12/20/2022] Open
Abstract
While gene transfer using recombinant adeno-associated viral (rAAV) vectors has shown success in some clinical trials, there remain many tissues that are not well transduced. Because of the recent success in reprogramming islet-derived cells into functional β cells in animal models, we constructed 2 highly complex barcoded replication competent capsid shuffled libraries and selected for high-transducing variants on primary human islets. We describe the generation of a chimeric AAV capsid (AAV-KP1) that facilitates transduction of primary human islet cells and human embryonic stem cell-derived β cells with up to 10-fold higher efficiency compared with previously studied best-in-class AAV vectors. Remarkably, this chimeric capsid also enabled transduction of both mouse and human hepatocytes at very high levels in a humanized chimeric mouse model, thus providing a versatile vector that has the potential to be used in both preclinical testing and human clinical trials for liver-based diseases and diabetes.
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Affiliation(s)
- Katja Pekrun
- Departments of Pediatrics and Genetics, Stanford University, Stanford, California, USA
| | - Gustavo De Alencastro
- Departments of Pediatrics and Genetics, Stanford University, Stanford, California, USA
| | - Qing-Jun Luo
- Departments of Pediatrics and Genetics, Stanford University, Stanford, California, USA
| | - Jun Liu
- Departments of Pediatrics and Genetics, Stanford University, Stanford, California, USA
| | - Youngjin Kim
- UCSF Diabetes Center, UCSF, San Francisco, California, USA
| | - Sean Nygaard
- Oregon Stem Cell Center, Oregon Health & Science University (OHSU), Portland, Oregon, USA
| | - Feorillo Galivo
- Oregon Stem Cell Center, Oregon Health & Science University (OHSU), Portland, Oregon, USA
| | - Feijie Zhang
- Departments of Pediatrics and Genetics, Stanford University, Stanford, California, USA
| | - Ren Song
- Departments of Pediatrics and Genetics, Stanford University, Stanford, California, USA
| | - Matthew R. Tiffany
- Departments of Pediatrics and Genetics, Stanford University, Stanford, California, USA
| | - Jianpeng Xu
- Departments of Pediatrics and Genetics, Stanford University, Stanford, California, USA
| | | | - Markus Grompe
- Oregon Stem Cell Center, Oregon Health & Science University (OHSU), Portland, Oregon, USA
| | - Mark A. Kay
- Departments of Pediatrics and Genetics, Stanford University, Stanford, California, USA
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28
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Mietzsch M, Pénzes JJ, Agbandje-McKenna M. Twenty-Five Years of Structural Parvovirology. Viruses 2019; 11:E362. [PMID: 31010002 PMCID: PMC6521121 DOI: 10.3390/v11040362] [Citation(s) in RCA: 110] [Impact Index Per Article: 18.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2019] [Revised: 04/10/2019] [Accepted: 04/11/2019] [Indexed: 12/13/2022] Open
Abstract
Parvoviruses, infecting vertebrates and invertebrates, are a family of single-stranded DNA viruses with small, non-enveloped capsids with T = 1 icosahedral symmetry. A quarter of a century after the first parvovirus capsid structure was published, approximately 100 additional structures have been analyzed. This first structure was that of Canine Parvovirus, and it initiated the practice of structure-to-function correlation for the family. Despite high diversity in the capsid viral protein (VP) sequence, the structural topologies of all parvoviral capsids are conserved. However, surface loops inserted between the core secondary structure elements vary in conformation that enables the assembly of unique capsid surface morphologies within individual genera. These variations enable each virus to establish host niches by allowing host receptor attachment, specific tissue tropism, and antigenic diversity. This review focuses on the diversity among the parvoviruses with respect to the transcriptional strategy of the encoded VPs, the advances in capsid structure-function annotation, and therapeutic developments facilitated by the available structures.
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Affiliation(s)
- Mario Mietzsch
- Department of Biochemistry and Molecular Biology, Center for Structural Biology, The McKnight Brain Institute, University of Florida, Gainesville, FL 32610, USA.
| | - Judit J Pénzes
- Department of Biochemistry and Molecular Biology, Center for Structural Biology, The McKnight Brain Institute, University of Florida, Gainesville, FL 32610, USA.
| | - Mavis Agbandje-McKenna
- Department of Biochemistry and Molecular Biology, Center for Structural Biology, The McKnight Brain Institute, University of Florida, Gainesville, FL 32610, USA.
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29
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Doshi BS, Arruda VR. Gene therapy for hemophilia: what does the future hold? Ther Adv Hematol 2018; 9:273-293. [PMID: 30210756 DOI: 10.1177/2040620718791933] [Citation(s) in RCA: 72] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2018] [Accepted: 07/09/2018] [Indexed: 01/19/2023] Open
Abstract
Recent phase I/II adeno-associated viral vector-mediated gene therapy clinical trials have reported remarkable success in ameliorating disease phenotype in hemophilia A and B. These trials, which highlight the challenges overcome through decades of preclinical and first in human clinical studies, have generated considerable excitement for patients and caregivers alike. Optimization of vector and transgene expression has significantly improved the ability to achieve therapeutic factor levels in these subjects. Long-term follow-up studies will guide standardization of the approach with respect to the combination of serotype, promoter, dose, and manufacturing processes and inform safety for inclusion of young patients. Certain limitations preclude universal applicability of gene therapy, including transient liver transaminase elevations due to the immune responses to vector capsids or as yet undefined mechanisms, underlying liver disease from iatrogenic viral hepatitis, and neutralizing antibodies to clotting factors. Integrating vectors show promising preclinical results, but manufacturing and safety concerns still remain. The prospect of gene editing for correction of the underlying mutation is on the horizon with considerable potential. Herein, we review the advances and limitations that have resulted in these recent successful clinical trials and outline avenues that will allow for broader applicability of gene therapy.
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Affiliation(s)
- Bhavya S Doshi
- Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA, USA
| | - Valder R Arruda
- Department of Pediatrics, The Children's Hospital of Philadelphia, 3501 Civic Center Blvd, 5056 Colket Translational Research Center, Philadelphia, PA 19104, USA
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30
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Sun S, Schaffer DV. Engineered viral vectors for functional interrogation, deconvolution, and manipulation of neural circuits. Curr Opin Neurobiol 2018; 50:163-170. [PMID: 29614429 PMCID: PMC5984719 DOI: 10.1016/j.conb.2017.12.011] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2017] [Revised: 11/27/2017] [Accepted: 12/16/2017] [Indexed: 12/19/2022]
Abstract
Optimization of traditional replication-competent viral tracers has granted access to immediate synaptic partners of target neuronal populations, enabling the dissection of complex brain circuits into functional neural pathways. The excessive virulence of most conventional tracers, however, impedes their utility in revealing and genetically perturbing cellular function on long time scales. As a promising alternative, the natural capacity of adeno-associated viral (AAV) vectors to safely mediate persistent and robust gene expression has stimulated strong interest in adapting them for sparse neuronal labeling and physiological studies. Furthermore, increasingly refined engineering strategies have yielded novel AAV variants with enhanced target specificity, transduction, and retrograde trafficking in the CNS. These potent vectors offer new opportunities for characterizing the identity and connectivity of single neurons within immense networks and modulating their activity via robust delivery of functional genetic tools.
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Affiliation(s)
- Sabrina Sun
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, USA
| | - David V Schaffer
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, USA; Department of Bioengineering, University of California, Berkeley, CA, USA; The Helen Wills Neuroscience Institute, University of California, Berkeley, CA, USA; Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
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31
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Xiao Q, Min T, Ma S, Hu L, Chen H, Lu D. Intracellular generation of single-strand template increases the knock-in efficiency by combining CRISPR/Cas9 with AAV. Mol Genet Genomics 2018; 293:1051-1060. [PMID: 29671068 DOI: 10.1007/s00438-018-1437-2] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2018] [Accepted: 04/04/2018] [Indexed: 11/30/2022]
Abstract
Targeted integration of transgenes facilitates functional genomic research and holds prospect for gene therapy. The established microhomology-mediated end-joining (MMEJ)-based strategy leads to the precise gene knock-in with easily constructed donor, yet the limited efficiency remains to be further improved. Here, we show that single-strand DNA (ssDNA) donor contributes to efficient increase of knock-in efficiency and establishes a method to achieve the intracellular linearization of long ssDNA donor. We identified that the CRISPR/Cas9 system is responsible for breaking double-strand DNA (dsDNA) of palindromic structure in inverted terminal repeats (ITRs) region of recombinant adeno-associated virus (AAV), leading to the inhibition of viral second-strand DNA synthesis. Combing Cas9 plasmids targeting genome and ITR with AAV donor delivery, the precise knock-in of gene cassette was achieved, with 13-14% of the donor insertion events being mediated by MMEJ in HEK 293T cells. This study describes a novel method to integrate large single-strand transgene cassettes into the genomes, increasing knock-in efficiency by 13.6-19.5-fold relative to conventional AAV-mediated method. It also provides a comprehensive solution to the challenges of complicated production and difficult delivery with large exogenous fragments.
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Affiliation(s)
- Qing Xiao
- State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, Shanghai, 200438, P. R. China
| | - Taishan Min
- State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, Shanghai, 200438, P. R. China
| | - Shuangping Ma
- State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, Shanghai, 200438, P. R. China
| | - Lingna Hu
- State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, Shanghai, 200438, P. R. China
| | - Hongyan Chen
- State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, Shanghai, 200438, P. R. China.
| | - Daru Lu
- State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, Shanghai, 200438, P. R. China.
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32
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Brown N, Song L, Kollu NR, Hirsch ML. Adeno-Associated Virus Vectors and Stem Cells: Friends or Foes? Hum Gene Ther 2018; 28:450-463. [PMID: 28490211 DOI: 10.1089/hum.2017.038] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
The infusion of healthy stem cells into a patient-termed "stem-cell therapy"-has shown great promise for the treatment of genetic and non-genetic diseases, including mucopolysaccharidosis type 1, Parkinson's disease, multiple sclerosis, numerous immunodeficiency disorders, and aplastic anemia. Stem cells for cell therapy can be collected from the patient (autologous) or collected from another "healthy" individual (allogeneic). The use of allogenic stem cells is accompanied with the potentially fatal risk that the transplanted donor T cells will reject the patient's cells-a process termed "graft-versus-host disease." Therefore, the use of autologous stem cells is preferred, at least from the immunological perspective. However, an obvious drawback is that inherently as "self," they contain the disease mutation. As such, autologous cells for use in cell therapies often require genetic "correction" (i.e., gene addition or editing) prior to cell infusion and therefore the requirement for some form of nucleic acid delivery, which sets the stage for the AAV controversy discussed herein. Despite being the most clinically applied gene delivery context to date, unlike other more concerning integrating and non-integrating vectors such as retroviruses and adenovirus, those based on adeno-associated virus (AAV) have not been employed in the clinic. Furthermore, published data regarding AAV vector transduction of stem cells are inconsistent in regards to vector transduction efficiency, while the pendulum swings far in the other direction with demonstrations of AAV vector-induced toxicity in undifferentiated cells. The variation present in the literature examining the transduction efficiency of AAV vectors in stem cells may be due to numerous factors, including inconsistencies in stem-cell collection, cell culture, vector preparation, and/or transduction conditions. This review summarizes the controversy surrounding AAV vector transduction of stem cells, hopefully setting the stage for future elucidation and eventual therapeutic applications.
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Affiliation(s)
- Nolan Brown
- 1 Gene Therapy Center, University of North Carolina at Chapel Hill , North Carolina.,2 Department of Ophthalmology, University of North Carolina at Chapel Hill , North Carolina
| | - Liujiang Song
- 1 Gene Therapy Center, University of North Carolina at Chapel Hill , North Carolina.,2 Department of Ophthalmology, University of North Carolina at Chapel Hill , North Carolina
| | - Nageswara R Kollu
- 1 Gene Therapy Center, University of North Carolina at Chapel Hill , North Carolina.,2 Department of Ophthalmology, University of North Carolina at Chapel Hill , North Carolina
| | - Matthew L Hirsch
- 1 Gene Therapy Center, University of North Carolina at Chapel Hill , North Carolina.,2 Department of Ophthalmology, University of North Carolina at Chapel Hill , North Carolina
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33
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Cachón-González MB, Zaccariotto E, Cox TM. Genetics and Therapies for GM2 Gangliosidosis. Curr Gene Ther 2018; 18:68-89. [PMID: 29618308 PMCID: PMC6040173 DOI: 10.2174/1566523218666180404162622] [Citation(s) in RCA: 71] [Impact Index Per Article: 10.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2017] [Revised: 01/10/2018] [Accepted: 01/27/2018] [Indexed: 12/30/2022]
Abstract
Tay-Sachs disease, caused by impaired β-N-acetylhexosaminidase activity, was the first GM2 gangliosidosis to be studied and one of the most severe and earliest lysosomal diseases to be described. The condition, associated with the pathological build-up of GM2 ganglioside, has acquired almost iconic status and serves as a paradigm in the study of lysosomal storage diseases. Inherited as a classical autosomal recessive disorder, this global disease of the nervous system induces developmental arrest with regression of attained milestones; neurodegeneration progresses rapidly to cause premature death in young children. There is no effective treatment beyond palliative care, and while the genetic basis of GM2 gangliosidosis is well established, the molecular and cellular events, from diseasecausing mutations and glycosphingolipid storage to disease manifestations, remain to be fully delineated. Several therapeutic approaches have been attempted in patients, including enzymatic augmentation, bone marrow transplantation, enzyme enhancement, and substrate reduction therapy. Hitherto, none of these stratagems has materially altered the course of the disease. Authentic animal models of GM2 gangliodidosis have facilitated in-depth evaluation of innovative applications such as gene transfer, which in contrast to other interventions, shows great promise. This review outlines current knowledge pertaining the pathobiology as well as potential innovative treatments for the GM2 gangliosidoses.
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Affiliation(s)
| | - Eva Zaccariotto
- Department of Medicine, University of Cambridge, Cambridge, UK
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34
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Gaj T, Staahl BT, Rodrigues GMC, Limsirichai P, Ekman FK, Doudna JA, Schaffer DV. Targeted gene knock-in by homology-directed genome editing using Cas9 ribonucleoprotein and AAV donor delivery. Nucleic Acids Res 2017; 45:e98. [PMID: 28334779 PMCID: PMC5499784 DOI: 10.1093/nar/gkx154] [Citation(s) in RCA: 69] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2016] [Accepted: 02/22/2017] [Indexed: 12/27/2022] Open
Abstract
Realizing the full potential of genome editing requires the development of efficient and broadly applicable methods for delivering programmable nucleases and donor templates for homology-directed repair (HDR). The RNA-guided Cas9 endonuclease can be introduced into cells as a purified protein in complex with a single guide RNA (sgRNA). Such ribonucleoproteins (RNPs) can facilitate the high-fidelity introduction of single-base substitutions via HDR following co-delivery with a single-stranded DNA oligonucleotide. However, combining RNPs with transgene-containing donor templates for targeted gene addition has proven challenging, which in turn has limited the capabilities of the RNP-mediated genome editing toolbox. Here, we demonstrate that combining RNP delivery with naturally recombinogenic adeno-associated virus (AAV) donor vectors enables site-specific gene insertion by homology-directed genome editing. Compared to conventional plasmid-based expression vectors and donor templates, we show that combining RNP and AAV donor delivery increases the efficiency of gene addition by up to 12-fold, enabling the creation of lineage reporters that can be used to track the conversion of striatal neurons from human fibroblasts in real time. These results thus illustrate the potential for unifying nuclease protein delivery with AAV donor vectors for homology-directed genome editing.
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Affiliation(s)
- Thomas Gaj
- Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA
| | - Brett T Staahl
- Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
| | - Gonçalo M C Rodrigues
- Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA.,Department of Bioengineering and Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisbon, Portugal
| | - Prajit Limsirichai
- Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA 94720, USA
| | - Freja K Ekman
- Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA
| | - Jennifer A Doudna
- Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.,Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA.,Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA.,MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
| | - David V Schaffer
- Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA.,Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, CA 94720, USA.,Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA 94720, USA
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35
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Grosse S, Penaud-Budloo M, Herrmann AK, Börner K, Fakhiri J, Laketa V, Krämer C, Wiedtke E, Gunkel M, Ménard L, Ayuso E, Grimm D. Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells. J Virol 2017; 91:e01198-17. [PMID: 28768875 PMCID: PMC5625497 DOI: 10.1128/jvi.01198-17] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2017] [Accepted: 07/28/2017] [Indexed: 12/16/2022] Open
Abstract
The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids.IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus-like particles composed solely of the major capsid protein VP3, AAP's role in and relevance for assembly of genuine AAV capsids have remained largely unclear. Thus, we established a trans-complementation assay permitting assessment of AAP functionality during production of recombinant vectors based on complete AAV capsids and derived from any serotype. We find that AAP is indeed a critical factor not only for AAV2, but also for generation of vectors derived from nine other AAV serotypes. Moreover, we identify a new role of AAP in maintaining capsid protein stability in mammalian and insect cells. Thereby, our study expands our current understanding of AAV/AAP biology, and it concomitantly provides insights into the importance of AAP for AAV vector production.
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Affiliation(s)
- Stefanie Grosse
- Department of Infectious Diseases/Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, Germany
- BioQuant Center, University of Heidelberg, Heidelberg, Germany
| | - Magalie Penaud-Budloo
- INSERM UMR1089, University of Nantes, Centre Hospitalier Universitaire, Nantes, France
| | - Anne-Kathrin Herrmann
- Department of Infectious Diseases/Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, Germany
- BioQuant Center, University of Heidelberg, Heidelberg, Germany
| | - Kathleen Börner
- Department of Infectious Diseases/Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, Germany
- BioQuant Center, University of Heidelberg, Heidelberg, Germany
- German Center for Infection Research, Partner Site Heidelberg, Heidelberg, Germany
| | - Julia Fakhiri
- Department of Infectious Diseases/Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, Germany
- BioQuant Center, University of Heidelberg, Heidelberg, Germany
| | - Vibor Laketa
- Department of Infectious Diseases/Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, Germany
- German Center for Infection Research, Partner Site Heidelberg, Heidelberg, Germany
| | - Chiara Krämer
- Department of Infectious Diseases/Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, Germany
- BioQuant Center, University of Heidelberg, Heidelberg, Germany
| | - Ellen Wiedtke
- Department of Infectious Diseases/Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, Germany
- BioQuant Center, University of Heidelberg, Heidelberg, Germany
| | - Manuel Gunkel
- BioQuant Center, University of Heidelberg, Heidelberg, Germany
- CellNetworks Advanced Biological Screening Facility, University of Heidelberg, Heidelberg, Germany
| | - Lucie Ménard
- INSERM UMR1089, University of Nantes, Centre Hospitalier Universitaire, Nantes, France
| | - Eduard Ayuso
- INSERM UMR1089, University of Nantes, Centre Hospitalier Universitaire, Nantes, France
| | - Dirk Grimm
- Department of Infectious Diseases/Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, Germany
- BioQuant Center, University of Heidelberg, Heidelberg, Germany
- German Center for Infection Research, Partner Site Heidelberg, Heidelberg, Germany
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36
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Chandrasekaran AP, Song M, Ramakrishna S. Genome editing: a robust technology for human stem cells. Cell Mol Life Sci 2017; 74:3335-3346. [PMID: 28405721 PMCID: PMC11107609 DOI: 10.1007/s00018-017-2522-0] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2016] [Revised: 04/05/2017] [Accepted: 04/07/2017] [Indexed: 12/20/2022]
Abstract
Human pluripotent stem cells comprise induced pluripotent and embryonic stem cells, which have tremendous potential for biological and therapeutic applications. The development of efficient technologies for the targeted genome alteration of stem cells in disease models is a prerequisite for utilizing stem cells to their full potential. Genome editing of stem cells is possible with the help of synthetic nucleases that facilitate site-specific modification of a gene of interest. Recent advances in genome editing techniques have improved the efficiency and speed of the development of stem cells for human disease models. Zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system are powerful tools for editing DNA at specific loci. Here, we discuss recent technological advances in genome editing with site-specific nucleases in human stem cells.
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Affiliation(s)
| | - Minjung Song
- Division of Bioindustry, Department of Food Biotechnology, College of Medical and Life Science, Silla University, Seoul, Republic of Korea.
| | - Suresh Ramakrishna
- Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, Republic of Korea.
- College of Medicine, Hanyang University, Seoul, Republic of Korea.
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37
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Osmon KJL, Woodley E, Thompson P, Ong K, Karumuthil-Melethil S, Keimel JG, Mark BL, Mahuran D, Gray SJ, Walia JS. Systemic Gene Transfer of a Hexosaminidase Variant Using an scAAV9.47 Vector Corrects GM2 Gangliosidosis in Sandhoff Mice. Hum Gene Ther 2017; 27:497-508. [PMID: 27199088 DOI: 10.1089/hum.2016.015] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
GM2 gangliosidosis is a group of neurodegenerative diseases caused by β-hexosaminidase A (HexA) enzyme deficiency. There is currently no cure. HexA is composed of two similar, nonidentical subunits, α and β, which must interact with the GM2 activator protein (GM2AP), a substrate-specific cofactor, to hydrolyze GM2 ganglioside. Mutations in either subunit or the activator can result in the accumulation of GM2 ganglioside within neurons throughout the central nervous system. The resulting neuronal cell death induces the primary symptoms of the disease: motor impairment, seizures, and sensory impairments. This study assesses the long-term effects of gene transfer in a Sandhoff (β-subunit knockout) mouse model. The study utilized a modified human β-hexosaminidase α-subunit (μ-subunit) that contains critical sequences from the β-subunit that enables formation of a stable homodimer (HexM) and interaction with GM2AP to hydrolyze GM2 ganglioside. We investigated a self-complementary adeno-associated viral (scAAV) vector expressing HexM, through intravenous injections of the neonatal mice. We monitored one cohort for 8 weeks and another cohort long-term for survival benefit, behavioral, biochemical, and molecular analyses. Untreated Sandhoff disease (SD) control mice reached a humane endpoint at approximately 15 weeks, whereas treated mice had a median survival age of 40 weeks, an approximate 2.5-fold survival advantage. On behavioral tests, the treated mice outperformed their knockout age-matched controls and perform similarly to the heterozygous controls. Through the enzymatic and GM2 ganglioside analyses, we observed a significant decrease in the GM2 ganglioside level, even though the enzyme levels were not significantly increased. Molecular analyses revealed a global distribution of the vector between brain and spinal cord regions. In conclusion, the neonatal delivery of a novel viral vector expressing the human HexM enzyme is effective in ameliorating the SD mouse phenotype for long-term. Our data could have implications not only for treatment of SD but also for Tay-Sachs disease (α-subunit deficiency) and similar brain disorders.
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Affiliation(s)
- Karlaina J L Osmon
- 1 Centre for Neuroscience Studies, Queen's University , Kingston, Ontario, Canada
| | - Evan Woodley
- 2 Department of Biomedical and Molecular Sciences, Queen's University , Kingston, Ontario, Canada
| | - Patrick Thompson
- 3 Medical Genetics/Departments of Pediatrics, Queen's University , Kingston, Ontario, Canada
| | - Katalina Ong
- 3 Medical Genetics/Departments of Pediatrics, Queen's University , Kingston, Ontario, Canada
| | | | - John G Keimel
- 5 New Hope Research Foundation , North Oaks, Minnesota
| | - Brian L Mark
- 6 Department of Microbiology, University of Manitoba , Winnipeg, Manitoba, Canada
| | - Don Mahuran
- 7 Genetics and Genome Biology, SickKids, Toronto, Ontario, Canada .,8 Department of Laboratory Medicine and Pathology, University of Toronto , Toronto, Ontario, Canada
| | - Steven J Gray
- 4 Gene Therapy Center, University of North Carolina , Chapel Hill, North Carolina.,9 Department of Ophthalmology, University of North Carolina , Chapel Hill, North Carolina
| | - Jagdeep S Walia
- 1 Centre for Neuroscience Studies, Queen's University , Kingston, Ontario, Canada .,2 Department of Biomedical and Molecular Sciences, Queen's University , Kingston, Ontario, Canada .,3 Medical Genetics/Departments of Pediatrics, Queen's University , Kingston, Ontario, Canada
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38
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Surface modulatable nanocapsids for targeting and tracking toward nanotheranostic delivery. Pharm Pat Anal 2017; 5:307-17. [PMID: 27610752 DOI: 10.4155/ppa-2016-0021] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Nanoparticle diagnostics and therapeutics (nanotheranostics) have significantly advanced cancer detection and treatment. However, many nanotheranostics are ineffective due to defects in tumor localization and bioavailability. An engineered Hepatitis E Virus (HEV) nanocapsid is a proposed platform for targeted cancer-cell delivery. Self-assembling from HEV capsid subunits, nanocapsids retain the capacity to enter cells and resist proteolytic/acidic conditions, but lack infectious viral elements. The nanocapsid surface was modified for chemical activation to confer tumor-specific targeting and detection, immune-response manipulation and controlled theranostic delivery. Nanotheranostic molecules can be packaged in the hollow nanocapsid shell during in vitro assembly. Complementing the adapted stability and cell-entry characteristics of the HEV capsid, a modified nanocapsid serves as a tunable tumor-targeting platform for nanotheronostic delivery.
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39
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Combining Engineered Nucleases with Adeno-associated Viral Vectors for Therapeutic Gene Editing. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2017; 1016:29-42. [PMID: 29130152 DOI: 10.1007/978-3-319-63904-8_2] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
With the recent advent of several generations of targeted DNA nucleases, most recently CRISPR/Cas9, genome editing has become broadly accessible across the biomedical community. Importantly, the capacity of these nucleases to modify specific genomic loci associated with human disease could render new classes of genetic disease, including autosomal dominant or even idiopathic disease, accessible to gene therapy. In parallel, the emergence of adeno-associated virus (AAV) as a clinically important vector raises the possibility of integrating these two technologies towards the development of gene editing therapies. Though clear challenges exist, numerous proof-of-concept studies in preclinical models offer exciting promise for the future of gene therapy.
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40
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Gaj T, Schaffer DV. Adeno-Associated Virus-Mediated Delivery of CRISPR-Cas Systems for Genome Engineering in Mammalian Cells. Cold Spring Harb Protoc 2016; 2016:2016/11/pdb.prot086868. [PMID: 27803249 DOI: 10.1101/pdb.prot086868] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Abstract
The CRISPR-Cas9 system has emerged as a highly versatile platform for introducing targeted genome modifications into mammalian cells and model organisms. However, fully capitalizing on the therapeutic potential for this system requires its safe and efficient delivery into relevant cell types. Adeno-associated virus (AAV) vectors are a clinically promising class of engineered gene-delivery vehicles capable of safely infecting a broad range of dividing and nondividing cell types, while also serving as a highly effective donor template for homology-directed repair. Together, CRISPR-Cas9 and AAV technologies have the potential to accelerate both basic research and clinical applications of genome engineering. Here, we present a step-by-step protocol for AAV-mediated delivery of CRISPR-Cas systems into mammalian cells. Procedures are given for the preparation of high-titer virus capable of achieving a diverse range of genetic modifications, including gene knockout and integration.
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Affiliation(s)
- Thomas Gaj
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California 94720
| | - David V Schaffer
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California 94720.,Department of Bioengineering, University of California, Berkeley, California 94720.,Department of Cell and Molecular Biology, University of California, Berkeley, California 94720.,Helen Wills Neuroscience Institute, University of California, Berkeley, California 94720
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41
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Santiago-Ortiz JL, Schaffer DV. Adeno-associated virus (AAV) vectors in cancer gene therapy. J Control Release 2016; 240:287-301. [PMID: 26796040 PMCID: PMC4940329 DOI: 10.1016/j.jconrel.2016.01.001] [Citation(s) in RCA: 148] [Impact Index Per Article: 16.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2015] [Revised: 12/08/2015] [Accepted: 01/02/2016] [Indexed: 02/06/2023]
Abstract
Gene delivery vectors based on adeno-associated virus (AAV) have been utilized in a large number of gene therapy clinical trials, which have demonstrated their strong safety profile and increasingly their therapeutic efficacy for treating monogenic diseases. For cancer applications, AAV vectors have been harnessed for delivery of an extensive repertoire of transgenes to preclinical models and, more recently, clinical trials involving certain cancers. This review describes the applications of AAV vectors to cancer models and presents developments in vector engineering and payload design aimed at tailoring AAV vectors for transduction and treatment of cancer cells. We also discuss the current status of AAV clinical development in oncology and future directions for AAV in this field.
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Affiliation(s)
- Jorge L Santiago-Ortiz
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, USA
| | - David V Schaffer
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, USA; Department of Bioengineering, University of California, Berkeley, CA, USA; Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA; The Helen Wills Neuroscience Institute, University of California, Berkeley, CA, USA.
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42
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Green DW, Watson GS, Watson JA, Lee DJ, Lee JM, Jung HS. Diversification and enrichment of clinical biomaterials inspired by Darwinian evolution. Acta Biomater 2016; 42:33-45. [PMID: 27381524 DOI: 10.1016/j.actbio.2016.06.039] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2016] [Revised: 06/11/2016] [Accepted: 06/21/2016] [Indexed: 02/06/2023]
Abstract
UNLABELLED Regenerative medicine and biomaterials design are driven by biomimicry. There is the essential requirement to emulate human cell, tissue, organ and physiological complexity to ensure long-lasting clinical success. Biomimicry projects for biomaterials innovation can be re-invigorated with evolutionary insights and perspectives, since Darwinian evolution is the original dynamic process for biological organisation and complexity. Many existing human inspired regenerative biomaterials (defined as a nature generated, nature derived and nature mimicking structure, produced within a biological system, which can deputise for, or replace human tissues for which it closely matches) are without important elements of biological complexity such as, hierarchy and autonomous actions. It is possible to engineer these essential elements into clinical biomaterials via bioinspired implementation of concepts, processes and mechanisms played out during Darwinian evolution; mechanisms such as, directed, computational, accelerated evolutions and artificial selection contrived in the laboratory. These dynamos for innovation can be used during biomaterials fabrication, but also to choose optimal designs in the regeneration process. Further evolutionary information can help at the design stage; gleaned from the historical evolution of material adaptations compared across phylogenies to changes in their environment and habitats. Taken together, harnessing evolutionary mechanisms and evolutionary pathways, leading to ideal adaptations, will eventually provide a new class of Darwinian and evolutionary biomaterials. This will provide bioengineers with a more diversified and more efficient innovation tool for biomaterial design, synthesis and function than currently achieved with synthetic materials chemistry programmes and rational based materials design approach, which require reasoned logic. It will also inject further creativity, diversity and richness into the biomedical technologies that we make. All of which are based on biological principles. Such evolution-inspired biomaterials have the potential to generate innovative solutions, which match with existing bioengineering problems, in vital areas of clinical materials translation that include tissue engineering, gene delivery, drug delivery, immunity modulation, and scar-less wound healing. STATEMENT OF SIGNIFICANCE Evolution by natural selection is a powerful generator of innovations in molecular, materials and structures. Man has influenced evolution for thousands of years, to create new breeds of farm animals and crop plants, but now molecular and materials can be molded in the same way. Biological molecules and simple structures can be evolved, literally in the laboratory. Furthermore, they are re-designed via lessons learnt from evolutionary history. Through a 3-step process to (1) create variants in material building blocks, (2) screen the variants with beneficial traits/properties and (3) select and support their self-assembly into usable materials, improvements in design and performance can emerge. By introducing biological molecules and small organisms into this process, it is possible to make increasingly diversified, sophisticated and clinically relevant materials for multiple roles in biomedicine.
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Affiliation(s)
- D W Green
- Division in Anatomy and Developmental Biology, Department of Oral Biology, Oral Science Research Center, BK21 PLUS Project, Yonsei University College of Dentistry, Seoul, Republic of Korea; Oral Biosciences, Faculty of Dentistry, The University of Hong Kong, 34, Hospital Road, Hong Kong SAR
| | - G S Watson
- School of Science & Engineering, University of the Sunshine Coast, Sippy Downs, QLD 4556, Australia
| | - J A Watson
- School of Science & Engineering, University of the Sunshine Coast, Sippy Downs, QLD 4556, Australia
| | - D-J Lee
- Division in Anatomy and Developmental Biology, Department of Oral Biology, Oral Science Research Center, BK21 PLUS Project, Yonsei University College of Dentistry, Seoul, Republic of Korea
| | - J-M Lee
- Division in Anatomy and Developmental Biology, Department of Oral Biology, Oral Science Research Center, BK21 PLUS Project, Yonsei University College of Dentistry, Seoul, Republic of Korea
| | - H-S Jung
- Division in Anatomy and Developmental Biology, Department of Oral Biology, Oral Science Research Center, BK21 PLUS Project, Yonsei University College of Dentistry, Seoul, Republic of Korea; Oral Biosciences, Faculty of Dentistry, The University of Hong Kong, 34, Hospital Road, Hong Kong SAR.
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43
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Kotterman MA, Chalberg TW, Schaffer DV. Viral Vectors for Gene Therapy: Translational and Clinical Outlook. Annu Rev Biomed Eng 2016; 17:63-89. [PMID: 26643018 DOI: 10.1146/annurev-bioeng-071813-104938] [Citation(s) in RCA: 319] [Impact Index Per Article: 35.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
In a range of human trials, viral vectors have emerged as safe and effective delivery vehicles for clinical gene therapy, particularly for monogenic recessive disorders, but there has also been early work on some idiopathic diseases. These successes have been enabled by research and development efforts focusing on vectors that combine low genotoxicity and immunogenicity with highly efficient delivery, including vehicles based on adeno-associated virus and lentivirus, which are increasingly enabling clinical success. However, numerous delivery challenges must be overcome to extend this success to many diseases; these challenges include developing techniques to evade preexisting immunity, to ensure more efficient transduction of therapeutically relevant cell types, to target delivery, and to ensure genomic maintenance. Fortunately, vector-engineering efforts are demonstrating promise in the development of next-generation gene therapy vectors that can overcome these barriers. This review highlights key historical trends in clinical gene therapy, the recent clinical successes of viral-based gene therapy, and current research that may enable future clinical application.
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Affiliation(s)
| | | | - David V Schaffer
- 4D Molecular Therapeutics, San Francisco, California 94107; .,University of California, Berkeley, California 94720-3220;
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44
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Hentzschel F, Herrmann AK, Mueller AK, Grimm D. Plasmodium meets AAV-the (un)likely marriage of parasitology and virology, and how to make the match. FEBS Lett 2016; 590:2027-45. [PMID: 27117587 DOI: 10.1002/1873-3468.12187] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2016] [Revised: 03/24/2016] [Accepted: 04/21/2016] [Indexed: 01/21/2023]
Abstract
The increasing use of screening technologies in malaria research has substantially expanded our knowledge on cellular factors hijacked by the Plasmodium parasite in the infected host, including those that participate in the clinically silent liver stage. This rapid gain in our understanding of the hepatic interaction partners now requires a means to validate and further disentangle parasite-host networks in physiologically relevant liver model systems. Here, we outline seminal work that contributed to our present knowledge on the intrahepatic Plasmodium host factors, followed by a discussion of surrogate models of mammalian livers or hepatocytes. We finally describe how Adeno-associated viruses could be engineered and used as hepatotropic tools to dissect Plasmodium-host interactions, and to deliberately control these networks for antimalaria vaccination or therapy.
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Affiliation(s)
- Franziska Hentzschel
- Department of Parasitology, Center for Infectious Diseases, Heidelberg University Hospital, Germany.,Department of Virology, Center for Infectious Diseases, Heidelberg University Hospital, Germany.,Cluster of Excellence CellNetworks, Heidelberg, Germany
| | - Anne-Kathrin Herrmann
- Department of Virology, Center for Infectious Diseases, Heidelberg University Hospital, Germany.,Cluster of Excellence CellNetworks, Heidelberg, Germany
| | - Ann-Kristin Mueller
- Department of Parasitology, Center for Infectious Diseases, Heidelberg University Hospital, Germany
| | - Dirk Grimm
- Department of Virology, Center for Infectious Diseases, Heidelberg University Hospital, Germany.,Cluster of Excellence CellNetworks, Heidelberg, Germany.,German Center for Infection Research (DZIF), Heidelberg, Germany
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45
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Maeder ML, Gersbach CA. Genome-editing Technologies for Gene and Cell Therapy. Mol Ther 2016; 24:430-46. [PMID: 26755333 PMCID: PMC4786923 DOI: 10.1038/mt.2016.10] [Citation(s) in RCA: 447] [Impact Index Per Article: 49.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2015] [Accepted: 01/07/2016] [Indexed: 12/11/2022] Open
Abstract
Gene therapy has historically been defined as the addition of new genes to human cells. However, the recent advent of genome-editing technologies has enabled a new paradigm in which the sequence of the human genome can be precisely manipulated to achieve a therapeutic effect. This includes the correction of mutations that cause disease, the addition of therapeutic genes to specific sites in the genome, and the removal of deleterious genes or genome sequences. This review presents the mechanisms of different genome-editing strategies and describes each of the common nuclease-based platforms, including zinc finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, and the CRISPR/Cas9 system. We then summarize the progress made in applying genome editing to various areas of gene and cell therapy, including antiviral strategies, immunotherapies, and the treatment of monogenic hereditary disorders. The current challenges and future prospects for genome editing as a transformative technology for gene and cell therapy are also discussed.
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Affiliation(s)
| | - Charles A Gersbach
- Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA
- Center for Genomic and Computational Biology, Duke University, Durham, North Carolina, USA
- Department of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina, USA
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46
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Yin PT, Han E, Lee KB. Engineering Stem Cells for Biomedical Applications. Adv Healthc Mater 2016; 5:10-55. [PMID: 25772134 PMCID: PMC5810416 DOI: 10.1002/adhm.201400842] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2014] [Revised: 02/14/2015] [Indexed: 12/19/2022]
Abstract
Stem cells are characterized by a number of useful properties, including their ability to migrate, differentiate, and secrete a variety of therapeutic molecules such as immunomodulatory factors. As such, numerous pre-clinical and clinical studies have utilized stem cell-based therapies and demonstrated their tremendous potential for the treatment of various human diseases and disorders. Recently, efforts have focused on engineering stem cells in order to further enhance their innate abilities as well as to confer them with new functionalities, which can then be used in various biomedical applications. These engineered stem cells can take on a number of forms. For instance, engineered stem cells encompass the genetic modification of stem cells as well as the use of stem cells for gene delivery, nanoparticle loading and delivery, and even small molecule drug delivery. The present Review gives an in-depth account of the current status of engineered stem cells, including potential cell sources, the most common methods used to engineer stem cells, and the utilization of engineered stem cells in various biomedical applications, with a particular focus on tissue regeneration, the treatment of immunodeficiency diseases, and cancer.
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Affiliation(s)
- Perry T Yin
- Department of Biomedical Engineering, Rutgers, The State University of New Jersey, 599 Taylor Road, Piscataway, NJ, 08854, USA
| | - Edward Han
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Toronto, ON, M5S 3G9, Canada
| | - Ki-Bum Lee
- Department of Biomedical Engineering, Rutgers, The State University of New Jersey, 599 Taylor Road, Piscataway, NJ, 08854, USA
- Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, 610 Taylor Road, Piscataway, NJ, 08854, USA
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47
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The Development and Use of Zinc-Finger Nucleases. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2016. [DOI: 10.1007/978-1-4939-3509-3_2] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
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48
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Mitchell AM, Moser R, Samulski RJ, Hirsch ML. Stimulation of AAV Gene Editing via DSB Repair. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2016. [DOI: 10.1007/978-1-4939-3509-3_8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/22/2022]
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Castle MJ, Turunen HT, Vandenberghe LH, Wolfe JH. Controlling AAV Tropism in the Nervous System with Natural and Engineered Capsids. Methods Mol Biol 2016; 1382:133-49. [PMID: 26611584 PMCID: PMC4993104 DOI: 10.1007/978-1-4939-3271-9_10] [Citation(s) in RCA: 97] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
More than one hundred naturally occurring variants of adeno-associated virus (AAV) have been identified, and this library has been further expanded by an array of techniques for modification of the viral capsid. AAV capsid variants possess unique antigenic profiles and demonstrate distinct cellular tropisms driven by differences in receptor binding. AAV capsids can be chemically modified to alter tropism, can be produced as hybrid vectors that combine the properties of multiple serotypes, and can carry peptide insertions that introduce novel receptor-binding activity. Furthermore, directed evolution of shuffled genome libraries can identify engineered variants with unique properties, and rational modification of the viral capsid can alter tropism, reduce blockage by neutralizing antibodies, or enhance transduction efficiency. This large number of AAV variants and engineered capsids provides a varied toolkit for gene delivery to the CNS and retina, with specialized vectors available for many applications, but selecting a capsid variant from the array of available vectors can be difficult. This chapter describes the unique properties of a range of AAV variants and engineered capsids, and provides a guide for selecting the appropriate vector for specific applications in the CNS and retina.
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Affiliation(s)
- Michael J Castle
- Research Institute of the Children's Hospital of Philadelphia, 502-G Abramson Pediatric Research Building, 3615 Civic Center Boulevard, Philadelphia, PA, 19104, USA
- Department of Neurosciences, University of California-San Diego, La Jolla, CA, 92093, USA
| | - Heikki T Turunen
- Department of Ophthalmology, Schepens Eye Research Institute, Massachusetts Eye and Ear Hospital, Harvard Medical School, Boston, MA, 02114, USA
| | - Luk H Vandenberghe
- Department of Ophthalmology, Schepens Eye Research Institute, Massachusetts Eye and Ear Hospital, Harvard Medical School, Boston, MA, 02114, USA
| | - John H Wolfe
- Research Institute of the Children's Hospital of Philadelphia, 502-G Abramson Pediatric Research Building, 3615 Civic Center Boulevard, Philadelphia, PA, 19104, USA.
- Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
- W.F. Goodman Center for Comparative Medical Genetics, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
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