Guang Qu

Reversal of Blindness in Animal Models of Leber Congenital Amaurosis Using Optimized AAV2-mediated Gene Transfer

We evaluated the safety and efficacy of an optimized adeno-associated virus (AAV; AAV2.RPE65) in animal models of the RPE65 form of Leber congenital amaurosis (LCA). Protein expression was optimized by addition of a modified Kozak sequence at the translational start site of hRPE65. Modifications in AAV production and delivery included use of a long stuffer sequence to prevent reverse packaging from the AAV inverted-terminal repeats, and co-injection with a surfactant. The latter allows consistent and predictable delivery of a given dose of vector. We observed improved electroretinograms (ERGs) and visual acuity in Rpe65 mutant mice. This has not been reported previously using AAV2 vectors. Subretinal delivery of 8.25 x 10(10) vector genomes in affected dogs was well tolerated both locally and systemically, and treated animals showed improved visual behavior and pupillary responses, and reduced nystagmus within 2 weeks of injection. ERG responses confirmed the reversal of visual deficit. Immunohistochemistry confirmed transduction of retinal pigment epithelium cells and there was minimal toxicity to the retina as judged by histopathologic analysis. The data demonstrate that AAV2.RPE65 delivers the RPE65 transgene efficiently and quickly to the appropriate target cells in vivo in animal models. This vector holds great promise for treatment of LCA due to RPE65 mutations.

Undetectable Transcription of cap in a Clinical AAV Vector: Implications for Preformed Capsid in Immune Responses

In a gene therapy clinical trial for hemophilia B, adeno-associated virus 2 (AAV2) capsid-specific CD8(+) T cells were previously implicated in the elimination of vector-transduced hepatocytes, resulting in loss of human factor IX (hFIX) transgene expression. To test the hypothesis that expression of AAV2 cap DNA impurities in the AAV2-hFIX vector was the source of epitopes presented on transduced cells, transcription of cap was assessed by quantitative reverse transcription-PCR (Q-RT-PCR) following transduction of target cells with the vector used in the clinical trial. Transcriptional profiling was also performed for residual Amp(R), and adenovirus E2A and E4. Although trace amounts of DNA impurities were present in the clinical vector, transcription of these sequences was not detected after transduction of human hepatocytes, nor in mice administered a dose 26-fold above the highest dose administered in the clinical study. Two methods used to minimize encapsidated DNA impurities in the clinical vector were: (i) a vector (cis) production plasmid with a backbone exceeding the packaging limit of AAV; and (ii) a vector purification step that achieved separation of the vector from vector-related impurities (e.g., empty capsids). In conclusion, residual cap expression was undetectable following transduction with AAV2-hFIX clinical vectors. Preformed capsid protein is implicated as the source of epitopes recognized by CD8(+) T cells that eliminated vector-transduced cells in the clinical study.

PEG-modulated column chromatography for purification of recombinant adeno-associated virus serotype 9.

Column chromatography has been described for purification of recombinant adeno-associated viral vectors (rAAV) serotypes 1, 2, 5, 6 and 8. Some of these purification processes have been used in manufacturing pre-clinical grade and clinical grade rAAV vectors. Recently, recombinant AAV9 has been reported to be highly efficient in transducing cardiac muscle in animal models. Systemic or cardiac gene delivery and other applications may require large quantities of rAAV9 vectors, thus a scalable method supporting large scale purification of rAAV9 is needed for clinical development. However, column chromatography-based purification has not been reported to date for rAAV9. This study reports a polyethylene glycol (PEG) modulated chromatography process for purification of AAV9 vectors. Inclusion of PEG in chromatography buffers modulated rAAV9 elution profiles in a manner that resulted in significantly improved resin binding capacity, vector purity and yield. PEG-modulated methods were developed and optimized for hydroxyapatite and ion exchange chromatography, and shown to result in vectors of high purity and functional activity.