Enhanced Transgene Expression from Recombinant Single-Stranded D-Sequence-Substituted Adeno-Associated Virus Vectors in Human Cell Lines In Vitro and in Murine Hepatocytes In Vivo

Abstract

The results of the studies described here not only have provided novel insights into some of the critical steps in the life cycle of a human virus, the adeno-associated virus (AAV), that causes no known disease but have also led to the development of novel recombinant AAV vectors which are more efficient in allowing increased levels of gene expression. Thus, these studies have significant implications for the potential use of these novel AAV vectors in human gene therapy.

Development of Optimized AAV Serotype Vectors for High-Efficiency Transduction at Further Reduced Doses

“…15 We have also described that for the genome-modified AAV2 vectors, LC1 and LC2, in which one of the D-sequences in the viral inverted terminal repeats (ITRs) was deleted, the efficiency of transgene expression from the single-stranded genomes was significantly increased. 17 Thus, as depicted schematically in Figure 1, we generated the following set of QMssAAV2 vectors containing the firefly luciferase (Fluc) reporter gene flanked by either the unmodified ITRs (QM-ssAAV2-WT-Fluc), or with D-sequence-deleted ITRs (QM-ssAAV2-LC1-Fluc and QM-ssAAV2-LC2-Fluc). A human hepatocellular carcinoma (HCC) cell line, Huh7, was transduced with each of the 3 optimized AAV2 vectors deliberately at a relative low MOI of 1000 vg/cell under identical conditions, and transgene expression was determined 72 hr posttransduction.…”

“…32 We have also described strategies involving genome modifications that lead to the generation of ssAAV vectors with which high efficiency of transgene expression can be achieved. 17 In the present studies, we combined the two strategies to generate the optimized AAV vectors. Reassuringly, the combination of the two strategies further augmented the transduction efficiency up to *6-fold, compared with that of the most efficient AAV2 and AAV3 serotype vectors containing single-stranded expression cassettes.…”

“…[14][15][16] In addition to the capsid-modified AAV vectors, we have also described the generation of genomemodified vectors, with which improved transgene expression from single-stranded AAV (ssAAV) vectors can be achieved. 17 In the present studies, we wished to examine whether combining the capsid modifications and genome modifications might lead to the generation of optimized AAV vectors capable of increased transduction at further reduced doses. To this end, ssAAV2 and ssAAV3 serotype vectors were generated using the strategy depicted schematically in Figure 1.…”

Adeno-Associated Virus: The Naturally Occurring Virus Versus the Recombinant Vector

“…[39][40][41] Another colleague, Giridhara Rao Jayandharan, PhD, and others identified the presence of a putative negative regulatory element in the D sequence in the 5¢ ITR, to which the NF-jB-repressing factor binds and inhibits transcription. 42 Yet another long-term colleague, Chen Ling, PhD, and others eventually developed one D sequence-deleted genome that led to the generation of the generation X (''GenX'') AAV2 vectors in 2015, 43 and documented significantly enhanced transgene expression in human cell lines in vitro as well as in murine hepatocytes in vivo.…”

Productive life cycle of adeno-associated virus serotype 2 in the complete absence of a conventional polyadenylation signal

“…Together, they facilitate replication and viral encapsidation of any DNA materials flanked by two ITRs and thus served as a positive control in our experiments. At various time points post-transfection, low-molecular-mass DNA samples were isolated, digested extensively with Dpn I to degrade input plasmids and subjected to Southern blot analysis as described previously (Ling et al, 2015). As seen in Fig.…”