Rational design of split gene vectors to expand the packaging capacity of adeno-associated viral vectors

"December 2007"The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file.Vita.Thesis (Ph. D.) University of Missouri-Columbia 2007.Adeno associated viruses (AAV) have recently been demonstrated as a very promising gene delivery vehicle. But the limited packaging capacity of AAV vectors (4̃.7kb) hinders their application for diseases involving large genes such as those responsible for Duchenne muscular dystrophy and cystic fibrosis. To overcome this hurdle, the trans-splicing and overlapping dual vector methods were developed to expand the packaging capacity of AAV. It has been demonstrated that certain expression limiting barriers affect transduction from these dual vectors. The trans-splicing method requires an optimal gene splitting site and the overlapping method requires a highly recombinogenic domain in the middle of the gene for high levels of transduction. To overcome these limitations of dual vectors, we developed a novel transsplicing/ overlapping hybrid vector system that can efficiently reconstitute any large gene. The experimental data demonstrate that the hybrid vector system improves gene expression compared to the traditional dual vectors. The study also demonstrates that the rationally designed transsplicing AAV vectors can be successfully used for body-wide gene delivery. Taken together, this study outlines the considerations to be taken into account for rational design of split gene vectors that would be capable of efficient transgene expression.Includes bibliographical reference

Improving the hybrid adeno-associated viral vectors with the exonic splicing enhancer

Abstract only availableAdeno-associated virus (AAV) is a rising champion for gene therapy due to its powerful and safe gene delivery properties. Yet the small packaging capacity of AAV limits its application for large therapeutic genes. The hybrid dual vectors show promise in overcoming this limitation (Ghosh 2008). In this approach, the gene of interest is split into 5' and 3' parts. Each part is packaged in a separate AAV virion. The 5' vector carries a splice donor, and the 3' vector a splice acceptor. Both also share an overlapping sequence. Recombination occurs simultaneously under two pathways, either through homologous recombination mediated by the overlapping sequence or head-to-tail ITR-mediated recombination. The recombined genome is spliced to generate mature mRNA. In an effort to further improve the hybrid vectors, we replaced the alkaline phosphatase (AP) overlapping sequence in a previously published LacZ hybrid vector set with an exonic splicing enhancer (ESE) sequence. The ESE sequences are known to increase splicing efficiency (Skordis, 2003). We hypothesized that the ESE sequence should increase splicing efficiency in the hybrid vectors, hence, lead to greater gene expression. To test this hypothesis, we transfected 293 cells with a series of different LacZ constructs. We then measured LacZ protein expression by cytochemical staining and enzymatic assay. Green fluorescent protein (GFP) containing plasmid was co-transfected as an internal control for transfection efficiency. The ESE-intron LacZ, unmodified intact LacZ, intron LacZ and AP-intron LacZ yielded β-galactosidase (LacZ) activity of 475 ± 36, 361 ± 18, 283 ± 9 and 225 ± 35 units/µg protein, respectively. Next, we split the ESE-intron LacZ gene into two separate constructs, which can be used to make recombinant AAV. Encouragingly, co-transfection of the split LacZ ESE-intron constructs resulted in 5.61 ± 0.76 units/µg protein β-galactosidase activity while the previously described split LacZ AP-intron and split LacZ intron constructs resulted in 3.93 ± 0.36 and 3.41 ± 0.36 units/µg protein β-galactosidase activity, respectively. Our results suggest that the ESE-intron vector is operating at par with the intact gene and may provide better gene delivery than the current generation of hybrid vectors. Future study with the corresponding recombinant AAV viruses may further reveal the utility of ESE containing hybrid vectors.Summer Research Internship in Cell & Molecular Biolog

AAV Serotype Influences Gene Transfer in Corneal Stroma \u3cem\u3ein vivo\u3c/em\u3e

This study evaluated the cellular tropism and relative transduction efficiency of three AAV serotypes, AAV6, AAV8 and AAV9, for corneal gene delivery using mouse cornea in vivo and donor human cornea ex vivo. The AAV6, AAV8 and AAV9 serotypes having AAV2 plasmid encoding for alkaline phosphatase (AP) gene were generated by transfecting HEK293 cell line with pHelper, pARAP4 and pRep/Cap plasmids. Viral vectors (109 vg/μl) were topically applied onto mouse cornea in vivo and human cornea ex vivo after removing the epithelium. Human corneas were processed for transgene delivery at day 5 after viral vector application. Mouse corneas were harvested at 4, 14 and 30 days after vector application for AP staining. Transduction efficiency was calculated by quantifying pixels of AP-stained area using Image J software and also confirmed by functional AP enzyme activity in the corneal lysates. Cellular toxicity of the three AAV serotypes was tested with TUNEL assay. Inflammatory response was detected by immunostaining for CD11b and F4/80. All three AAV serotypes successfully transduced mouse and human corneas. The order of transduction efficiency was AAV9\u3eAAV8\u3eAAV6. The transduction efficiency of AAV9 was 1.1–1.4 fold higher (p\u3e0.05) as compared to AAV8 and 3.5–5.5 fold higher (p\u3c0.01) as compared to AAV6. The level of transgene expression for all the three serotypes was greater at 14 days compared to 4 days and this high level of transgene expression was maintained up to the tested time point of 30 days. Corneas exposed to any of the three AAV serotypes did not show significant TUNEL positive cells or any inflammatory response as tested by CD11b or F4/80 staining suggesting that tested AAV serotypes do not induce cell death or inflammation and are safe for corneal gene therapy

Transduction Efficiency of AAV 2/6, 2/8 and 2/9 Vectors for Delivering Genes in Human Corneal Fibroblasts

In the present study, cellular tropism and relative transduction efficiency of AAV2/6, AAV2/8 and AAV2/9 vectors have been tested for the cornea using primary cultures of human corneal fibroblasts. The AAV6, AAV8 and AAV9 serotypes having AAV2 ITR plasmid encoding for alkaline phosphatase (AP) gene were generated by transfecting HEK293 cell line with pHelper, pARAP4 and pRep/Cap plasmids. Primary cultures of human corneal fibroblasts were exposed to AAV infectious particles at two different doses (1×105 and 2×105 MOI). Cytochemistry and enzyme assays were used to measure delivered transgene expression in samples collected at 4 and 30 hours after AAV infection by counting AP-stained cells or quantifying AP enzyme activity. Cellular toxicity of AAVs was evaluated with TUNEL and trypan blue assays. All three AAV serotypes transduced human corneal fibroblasts. The order of transduction efficiency was AAV2/6\u3e\u3e\u3eAAV2/9\u3eAAV2/8. The transduction efficiency of AAV2/6 was 30-50 fold higher (p \u3c0.001) for the human corneal fibroblasts compared to the AAV2/8 or AAV2/9 at two tested doses. The level of transgene expression at 4 hrs was considerably low compared to 30 hrs suggesting that the transgene delivery did not reach its peak at 4 hrs. Cultures exposed to any of the three AAV serotypes showed more than 97% cellular viability and less than 5 TUNEL positive cells suggesting that tested AAV serotypes do not induce significant cell death and are safe for corneal gene therapy

Tear biomarkers for keratoconus

Keratoconus is a progressive corneal thinning, ectatic condition, which affects vision. Recent advances in corneal topography measurements has helped advance proper diagnosis of this condition and increased research and clinical interests in the disease etiopathogenesis. Considerable progress has been achieved in understanding the progression of the disease and tear fluid has played a major role in the progress. This review discusses the importance of tear fluid as a source of biomarker for keratoconus and how advances in technology have helped map the complexity of tears and thereby molecular readouts of the disease. Expanding knowledge of the tear proteome, lipidome and metabolome opened up new avenues to study keratoconus and to identify probable prognostic or diagnostic biomarkers for the disease. A multidimensional approach of analyzing tear fluid of patients layering on proteomics, lipidomics and metabolomics is necessary in effectively decoding keratoconus and thereby identifying targets for its treatment

Targeted AAV5-Smad7 Gene Therapy Inhibits Corneal Scarring \u3cem\u3ein vivo\u3c/em\u3e

Corneal scarring is due to aberrant activity of the transforming growth factor β (TGFβ) signaling pathway following traumatic, mechanical, infectious, or surgical injury. Altered TGFβ signaling cascade leads to downstream Smad (Suppressor of mothers against decapentaplegic) protein-mediated signaling events that regulate expression of extracellular matrix and myogenic proteins. These events lead to transdifferentiation of keratocytes into myofibroblasts through fibroblasts and often results in permanent corneal scarring. Hence, therapeutic targets that reduce transdifferentiation of fibroblasts into myofibroblasts may provide a clinically relevant approach to treat corneal fibrosis and improve long-term visual outcomes. Smad7 protein regulates the functional effects of TGFβ signaling during corneal wound healing. We tested that targeted delivery of Smad7 using recombinant adeno-associated virus serotype 5 (AAV5-Smad7) delivered to the corneal stroma can inhibit corneal haze post photorefractive keratectomy (PRK) in vivo in a rabbit corneal injury model. We demonstrate that a single topical application of AAV5-Smad7 in rabbit cornea post-PRK led to a significant decrease in corneal haze and corneal fibrosis. Further, histopathology revealed lack of immune cell infiltration following AAV5-Smad7 gene transfer into the corneal stroma. Our data demonstrates that AAV5-Smad7 gene therapy is relatively safe with significant potential for the treatment of corneal disease currently resulting in fibrosis and impaired vision

Targeted AAV5-Smad7 gene therapy inhibits corneal scarring in vivo.

Corneal scarring is due to aberrant activity of the transforming growth factor β (TGFβ) signaling pathway following traumatic, mechanical, infectious, or surgical injury. Altered TGFβ signaling cascade leads to downstream Smad (Suppressor of mothers against decapentaplegic) protein-mediated signaling events that regulate expression of extracellular matrix and myogenic proteins. These events lead to transdifferentiation of keratocytes into myofibroblasts through fibroblasts and often results in permanent corneal scarring. Hence, therapeutic targets that reduce transdifferentiation of fibroblasts into myofibroblasts may provide a clinically relevant approach to treat corneal fibrosis and improve long-term visual outcomes. Smad7 protein regulates the functional effects of TGFβ signaling during corneal wound healing. We tested that targeted delivery of Smad7 using recombinant adeno-associated virus serotype 5 (AAV5-Smad7) delivered to the corneal stroma can inhibit corneal haze post photorefractive keratectomy (PRK) in vivo in a rabbit corneal injury model. We demonstrate that a single topical application of AAV5-Smad7 in rabbit cornea post-PRK led to a significant decrease in corneal haze and corneal fibrosis. Further, histopathology revealed lack of immune cell infiltration following AAV5-Smad7 gene transfer into the corneal stroma. Our data demonstrates that AAV5-Smad7 gene therapy is relatively safe with significant potential for the treatment of corneal disease currently resulting in fibrosis and impaired vision