Liver Transduction with Recombinant Adeno-Associated Virus Is Primarily Restricted by Capsid Serotype Not Vector Genotype

Abstract

We and others have recently reported highly efficient liver gene transfer with adeno-associated virus 8 (AAV-8) pseudotypes, i.e., AAV-2 genomes packaged into AAV-8 capsids. Here we studied whether liver transduction could be further enhanced by using viral DNA packaging sequences (inverted terminal repeats [ITRs]) derived from AAV genotypes other than 2. To this end, we generated two sets of vector constructs carrying expression cassettes embedding a gfp gene or the human factor IX (hfIX) gene flanked by ITRs from AAV genotypes 1 through 6. Initial in vitro analyses of gfp vector DNA replication, encapsidation, and cell transduction revealed a surprisingly high degree of interchangeability among the six genotypes. For subsequent in vivo studies, we cross-packaged the six hfIX variants into AAV-8 and infused mice via the portal vein with doses of 5 ؋ 10 10 to 1.8 ؋ 10 12 particles. Notably, all vectors expressed comparably high plasma hFIX levels within a dose cohort over the following 6 months, concurrent with the finding of equivalent vector DNA copy numbers per cell. Partial hepatectomies resulted in ϳ80% drops of hFIX levels and vector DNA copy numbers in all groups, indicating genotype-independent persistence of predominantly episomal vector DNA. Southern blot analyses of total liver DNA in fact confirmed the presence of identical and mostly nonintegrated molecular vector forms for all genotypes. We conclude that, unlike serotypes, AAV genotypes are not critical for efficient hepatocyte transduction and can be freely substituted. This corroborates our current model for AAV vector persistence in the liver and provides useful information for the future design and application of recombinant AAV.Gene transfer vectors based on the single-stranded DNA parvovirus AAV (adeno-associated virus) are enormously popular and powerful tools for in vivo delivery of small DNA expression cassettes. The list of human genes carried by such cassettes is growing steadily and spans a wealth of clinically relevant candidates expressing blood coagulation factors (28), the cystic fibrosis transmembrane conductance regulator (18), or dystrophin (32), among many others. Most recently, AAV vectors have begun to attract particular attention for delivery of short hairpin RNAs, and indeed, with a packaging capacity of 5 kb DNA and the promise to mediate safe and persistent gene transfer, they appear as bona fide tools for in vivo RNA interference applications (27).A major reason for AAV's broad and growing appeal is the feasibility to pseudotype the recombinant viral DNA, i.e., to generate particles in which the vector DNA (genotype) and the viral capsid (serotype) differ in their AAV origins (23). The resulting hybrid particles are typically characterized by unique receptor tropisms and are distinctly recognized by the host immune system, as determined by the capsid. Thus, the pseudotyping approach has dramatically extended the array of cells and tissues susceptible to AAV gene transfer and relieved some of the concerns associated with t...

In vivo post-transcriptional gene silencing of α-1 antitrypsin by adeno-associated virus vectors expressing siRNA

“…A high percentage of hepatocytes showed immunohistochemical evidence of a decrease in intracellular Z-AAT, which is consistent with earlier studies by a number of groups, indicating that rAAV8-pseudotyped vectors are quite efficient for transduction of murine liver. 19,28,41,42 In fact, the inability of the original rAAV2-based vectors Software, sections for each rAAV8-siRNA transduced livers were analyzed. Thresholded area represents the area on the section that is within the parameters considered to be positive staining.…”

“…19,[24][25][26][27][28] In this report, a number of small-interfering RNA (siRNA) constructs directed against AAT were expressed from rAAV vectors in vitro and in vivo and their ability to decrease the levels of intracellular Z-AAT within the liver was assessed. A tri-functional (3X) construct emerged from these studies as a potential candidate for future studies of gene therapy of AAT liver disease.…”

It's all about the clothing: capsid domination in the adeno‐associated viral vector world

“…However, attempts by Kay and colleagues to improve liver-directed AAV gene transfer efficiency by modifying the vector DNA (i.e. ITRs) among serotypes AAV1 to AAV6 were unsuccessful, [3] whereas switching vector capsid significantly influences vector performance [2,4,5].Cumulative evidence from preclinical and clinical studies using AAV vectors has demonstrated that the vector capsid is not only critical in determining the tropism and transduction efficiency, but also is a major antigen whose interaction with host immune responses will determine both the ability of the vector to access the target tissue and the resulting duration of expression.The work by VandenDriessche and colleagues in this issue of the Journal [6], together with recent reports [7][8][9], characterizes a new player in the AAV arena: AAV9 isolated from human tissues, another vivid example of what a capsid change can achieve in gene transfer experiments. These authors demonstrate that, similar to AAV8, AAV9 is effective in transducing hepatocytes of hemophilia B mice, which resulted in supraphysiological levels of biologically active factor (F) IX.…”

Potential of AAV vectors in the treatment of metabolic disease

“…Studies in adult mice have shown that partial hepatectomy, which is associated with a burst of hepatocellular proliferation, leads to rapid loss of episomal vector genomes. [14][15][16] Essentially the same phenomenon is seen following vector delivery to neonatal mice, where high rates of hepatocellular proliferation result in almost complete clearance of episomal vector genomes within two doublings of liver mass occurring within the first 1-2 weeks of life. 17,18 A stable basal level of transgene expression, probably reflecting vector integration, persists thereafter in up to 8% of hepatocytes depending on the vector dose used.…”