High-Efficiency Transduction of Primary Human Hematopoietic Stem/Progenitor Cells by AAV6 Vectors: Strategies for Overcoming Donor-Variation and Implications in Genome Editing

Chen, Ling; Bhukhai, Kanit; Yin, Zifei; Ming, Tan; Yoder, Mervin C.; Leboulch, Philippe; Payen, Emmanuel; Srivastava, Arun

doi:10.1038/srep35495

Cited by 28 publications

(36 citation statements)

References 37 publications

(31 reference statements)

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“…Recently, AAV6 has been used in conjunction with nucleasebased genome editing platforms for the delivery of donor DNA for homology-dependent repair of double-stranded DNA breaks (72)(73)(74)(75)(76). While AAV6-mediated donor delivery was efficient and editing was observed following the creation of DNA breaks, no AAV6-mediated editing was observed in the absence of nucleases (72)(73)(74)(75), in concordance with our findings.…”

Section: Discussionsupporting

confidence: 90%

Stem cell-derived clade F AAVs mediate high-efficiency homologous recombination-based genome editing

Smith¹,

Wright²,

Clark³

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

100

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The precise correction of genetic mutations at the nucleotide level is an attractive permanent therapeutic strategy for human disease. However, despite significant progress, challenges to efficient and accurate genome editing persist. Here, we report a genome editing platform based upon a class of hematopoietic stem cell (HSC)-derived clade F adeno-associated virus (AAV), which does not require prior nuclease-mediated DNA breaks and functions exclusively through BRCA2-dependent homologous recombination. Genome editing is guided by complementary homology arms and is highly accurate and seamless, with no evidence of on-target mutations, including insertion/deletions or inclusion of AAV inverted terminal repeats. Efficient genome editing was demonstrated at different loci within the human genome, including a safe harbor locus, AAVS1, and the therapeutically relevant IL2RG gene, and at the murine Rosa26 locus. HSC-derived AAV vector (AAVHSC)-mediated genome editing was robust in primary human cells, including CD34 cells, adult liver, hepatic endothelial cells, and myocytes. Importantly, high-efficiency gene editing was achieved in vivo upon a single i.v. injection of AAVHSC editing vectors in mice. Thus, clade F AAV-mediated genome editing represents a promising, highly efficient, precise, single-component approach that enables the development of therapeutic in vivo genome editing for the treatment of a multitude of human gene-based diseases.

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Section: Discussionsupporting

confidence: 90%

Stem cell-derived clade F AAVs mediate high-efficiency homologous recombination-based genome editing

Smith¹,

Wright²,

Clark³

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

100

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“…However, AAV1, modified AAV6, and AAV7 vectors have been found to efficiently transduce HSCs. 54,57,110,111 Furthermore, we show here that AAV8, AAV9, and AAV10 serotypes promote efficient thymocyte transduction. Although the properties of AAV vector genomes to exist primarily as episomes would be expected to negatively affect their ability to be used for stable gene therapy in dividing cells, long-term persistence of transgene expression has been detected in HSCs.…”

Section: Discussionsupporting

confidence: 53%

Intrathymic adeno-associated virus gene transfer rapidly restores thymic function and long-term persistence of gene-corrected T cells

Pouzolles

Machado

Guilbaud

et al. 2020

Journal of Allergy and Clinical Immunology

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Background: Patients with T-cell immunodeficiencies are generally treated with allogeneic hematopoietic stem cell transplantation, but alternatives are needed for patients without matched donors. An innovative intrathymic gene therapy approach that directly targets the thymus might improve outcomes. Objective: We sought to determine the efficacy of intrathymic adeno-associated virus (AAV) serotypes to transduce thymocyte subsets and correct the T-cell immunodeficiency in a zetaassociated protein of 70 kDa (ZAP-70)-deficient murine model. Methods: AAV serotypes were injected intrathymically into wild-type mice, and gene transfer efficiency was monitored. ZAP-70 2/2 mice were intrathymically injected with an AAV8 vector harboring the ZAP70 gene. Thymus structure, immunophenotyping, T-cell receptor clonotypes, T-cell function, immune responses to transgenes and autoantibodies, vector copy number, and integration were evaluated. Results: AAV8, AAV9, and AAV10 serotypes all transduced thymocyte subsets after in situ gene transfer, with transduction of up to 5% of cells. Intrathymic injection of an AAV8-ZAP-70 vector into ZAP-70 2/2 mice resulted in a rapid thymocyte differentiation associated with the development of a thymic medulla. Strikingly, medullary thymic epithelial cells expressing the autoimmune regulator were detected within 10 days of gene transfer, correlating with the presence of functional effector and regulatory T-cell subsets with diverse T-cell receptor clonotypes in the periphery. Although thymocyte reconstitution was transient, gene-corrected peripheral T cells harboring approximately 1 AAV genome per cell persisted for more than 40 weeks, and AAV vector integration was detected. Conclusions: Intrathymic AAV-transduced progenitors promote a rapid restoration of the thymic architecture, with a single wave of thymopoiesis generating long-term peripheral T-cell function.

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“…Recently, Dr Srivastava's group generated a triple-mutant (Y705F + Y731F + T492V) AAV6 vector by mutagenizing the surface-exposed Y and T residues on capsid proteins. 12,13 Combined with the strategy of ex vivo transduction at high cell density, the transduction efficiency can reach 90%. In the present study, we wish to uncover the underlying molecular mechanism to enhance rAAV6 transduction in HSPCs.…”

Section: Discussionmentioning

confidence: 99%

Mechanism for enhanced transduction of hematopoietic cells by recombinant adeno‐associated virus serotype 6 vectors

Chen

Jia

et al. 2020

FASEB j.

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Hematopoietic gene delivery, such as hematopoietic stem/progenitor cells (HSPCs), is a promising treatment for both inherited and acquired diseases, such as hemophilia. Recently, a combined strategy to achieve more than 90% transduction efficiency was documented using recombinant adeno‐associated virus serotype 6 (rAAV6) vectors. However, the mechanisms of enhanced vector transduction efficiency in hematopoietic cells are largely unknown. In this manuscript, we first reported that proteasome inhibitors, which are well‐known to facilitate rAAV intracellular trafficking in various cell types, are not effective in hematopoietic cells. From the screening of small molecules derived from traditional Chinese medicine, we demonstrated that shikonin, a potential reactive oxygen species (ROS) generator, significantly increased the in vitro and ex vivo transgene expression mediated by rAAV6 vectors in hematopoietic cells, including human cord blood‐derived CD34 + HSPCs. Shikonin mainly targeted vector intracellular trafficking, instead of host cell entry or endonuclear single to double strand vector DNA transition, in a vector serotype‐dependent manner. Moreover, a ROS scavenger completely prevented the capability of shikonin to enhance rAAV6 vector‐mediated transgene expression. Taken together, these studies expand our understanding of rAAV6‐mediated transduction in hematopoietic cells and are informative for improving rAAV6‐based treatment of blood diseases.

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High-Efficiency Transduction of Primary Human Hematopoietic Stem/Progenitor Cells by AAV6 Vectors: Strategies for Overcoming Donor-Variation and Implications in Genome Editing

Cited by 28 publications

References 37 publications

Stem cell-derived clade F AAVs mediate high-efficiency homologous recombination-based genome editing

Stem cell-derived clade F AAVs mediate high-efficiency homologous recombination-based genome editing

Intrathymic adeno-associated virus gene transfer rapidly restores thymic function and long-term persistence of gene-corrected T cells

Mechanism for enhanced transduction of hematopoietic cells by recombinant adeno‐associated virus serotype 6 vectors

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