Self-Complementary Adeno-Associated Virus Vectors Improve Transduction Efficiency of Corneal Endothelial Cells

Abstract

Transplantation of a donor cornea to restore vision is the most frequently performed transplantation in the world. Corneal endothelial cells (CEC) are crucial for the outcome of a graft as they maintain corneal transparency and avoid graft failure due to corneal opaqueness. Given the characteristic of being a monolayer and in direct contact with culture medium during cultivation in eye banks, CEC are specifically suitable for gene therapeutic approaches prior to transplantation. Recombinant adeno-associated virus 2 (rAAV2) vectors represent a promising tool for gene therapy of CEC. However, high vector titers are needed to achieve sufficient gene expression. One of the rate-limiting steps for transgene expression is the conversion of single-stranded (ss-) DNA vector genome into double-stranded (ds-) DNA. This step can be bypassed by using self-complementary (sc-) AAV2 vectors. Aim of this study was to compare for the first time transduction efficiencies of ss- and scAAV2 vectors in CEC. For this purpose AAV2 vectors containing enhanced green fluorescent protein (GFP) as transgene were used. Both in CEC and in donor corneas, transduction with scAAV2 resulted in significantly higher transgene expression compared to ssAAV2. The difference in transduction efficiency decreased with increasing vector titer. In most cases, only half the vector titer of scAAV2 was required for equal or higher gene expression rates than those of ssAAV2. In human donor corneas, GFP expression was 64.7±11.3% (scAAV) and 38.0±8.6% (ssAAV) (p<0.001), respectively. Furthermore, transduced cells maintained their viability and showed regular morphology. Working together with regulatory authorities, a translation of AAV2 vector-mediated gene therapy to achieve a temporary protection of corneal allografts during cultivation and transplantation could therefore become more realistic.

Advancements in the design and scalable production of viral gene transfer vectors

“…These libraries can be screened using high thoughput techniques for novel vector properties Self-complimentary vector backbones are designed to form an intramolecular dsDNA duplex, thus circumventing the need for de novo second-strand synthesis (Figure 4b). These vectors show stronger transcription and higher transduction efficiency compared to ssAAV vectors (Gruenert et al, 2016;McCarty, 2008;McCarty et al, 2001). Earlier reports of 140-fold increases in transduction efficiency are probably an artifact of a more rapid onset of gene expression in scAAV vectors at early time points.…”

“…This process is also vital in evading host antiviral defense mechanisms that recognize and degrade ssDNA (Schwartz et al, 2007). These vectors show stronger transcription and higher transduction efficiency compared to ssAAV vectors (Gruenert et al, 2016;McCarty, 2008;McCarty et al, 2001). Wild-type AAV contains just two ORF's, CAP and REP, as well as flanking inverted terminal repeat (ITR) sequences.…”

Gene therapy and the adeno‐associated virus in the treatment of genetic and acquired ophthalmic diseases in humans: Trials, future directions and safety considerations

“…In a study performed to look at transduction of corneal endothelial cells, it was found that scAAV vectors were more efficient in transducing cells when compared to the normal single‐stranded AAV (ssAAV) vectors. The study also showed that about half the scAAV vector titre was required to obtain a similar or higher transgene expression when compared to that of the ssAAV . One disadvantage of scAAV vectors is that the packaging limit is half of the traditional ssAAV vectors, highlighting the need for vectors that can package larger genes.…”

“…The study also showed that about half the scAAV vector titre was required to obtain a similar or higher transgene expression when compared to that of the ssAAV. 114 One disadvantage of scAAV vectors is that the packaging limit is half of the traditional ssAAV vectors, 115 highlighting the need for vectors that can package larger genes.…”

Clinical advances in gene therapy updates on clinical trials of gene therapy in haemophilia

“…The self‐complementary AAV (scAAV) vectors are designed as a single‐stranded inverted repeat, which folds back upon itself to form a double‐stranded (ds) genome when entering into infected cells. Therefore, the two half of a scAAV genome are complementary . In addition, the vector was designed to have FIX modifying transcriptional control elements (enhancer, promoter and polyadenylation site) to minimize size and allow packaging in the AAV vector; F9 cDNA was codon‐optimized to ameliorate the expression of the molecule .…”