AAV-mediated liver-directed gene therapy

Abstract

The liver is directly or indirectly involved in many essential processes and is affected by numerous inherited diseases. Therefore, many inherited diseases could be effectively treated by targeting the liver using gene transfer approaches. The challenges associated with liver-directed gene therapy are efficient targeting of hepatocytes, stability of the vector genome, and persistent high level expression. Many of these obstacles can be overcome with adeno-associated viral (AAV) gene transfer vectors. The first AAV gene transfer -vector developed for in vivo use was based on the AAV2 serotype. AAV2 has a broad tropism and transduces many cell types, including hepatocytes, relatively efficiently in vivo. The capsid protein confers the serological profile and at least 12 primate AAV serotypes have already been characterized. Importantly, pseudotyping a recombinant AAV vector with different capsid proteins can dramatically alter the tropism. Both AAV8 and AAV9 have higher affinities for hepatocytes when compared to AAV2. In particular, AAV8 can transduce three- to fourfold more hepatocytes and deliver three- to fourfold more genomes per transduced cell when compared to AAV2. Depending on the dose, AAV8 can transduce up to 90-95% of hepatocytes in the mouse liver following intraportal vein injection. Interestingly, comparable levels of transduction can be achieved following intravenous injection. Direct intraparenchymal injection of an AAV vector also mediates relatively high level long term expression. Additional specificity can be conferred by using liver-specific promoters in conjunction with AAV8 capsid proteins. In addition to treating primary hepatocyte defects, immune reactions to transgene products can be minimized by circumventing the fixed tissue macrophages of the liver, Kupffer cells, and limiting expression to hepatocytes. The ability to target hepatocytes by virtue of the AAV serotype and the use of liver-specific promoters allows investigators to test novel therapeutic approaches and answer basic clinical and biological questions.

Figure 1

Transduced viral cDNA persists in most tissues for at least 1 year. Tissues from 1-year-old mice were analyzed for the presence of viral cDNA using primers which amplify a 240 bp band from human cDNA, and a 454 bp band from the endogenous murine GUSB gene. A murine fibroblast line (A7) which has been transduced with a single retroviral copy of the human GUSB cDNA is shown as a positive control. Persistence of AAV-tranduced human GUSB cDNA is seen in most tissues at 1 year. The spleen, however, shows no indication of persistent viral transduction at this late time-point. The Southern blot shows the PCR products from a single mouse and is representative of the pattern observed in three separate mice 1 year after injection. Reproduced from Gene Therapy, 2001, with permission from Nature Publishing Group.

Figure 2

Comparison of efficiency of rAAV8-mediated liver transduction between tail vein and portal vein injections. (A) Plasma human coagulation factor IX (hF.IX) levels after tail vein (TV) or portal vein (PV) injection of AAV8-hF.IX16 into male C57BL/6 mice. Robust human coagulation factor IX expression with no lag phase was observed with both routes. Expression peaked 4 weeks after injection, followed by a substantial ( 75%) decline. Vertical bars indicate standard deviations. (B) Vector genome copy numbers (ds-vg/dge) in livers transduced with AAV8-EF1 -nlslacZ via tail vein or portal vein injection at 3.0 × 10¹¹ or 7.2 × 10¹² vg/mouse. Total liver DNA was extracted 6 weeks postinjection, and 10 μg of DNA was analyzed by Southern blot with BglI digestion and a 2.1-kb lacZ probe (BglI-BglI fragment). The left and right blots were analyzed separately with a different series of vector copy number standards. The double-stranded vector copy number standards (0 to 100 and 0 to 1,000 ds-vg/dge) were prepared by adding the corresponding amount of plasmid, pAAV-EF1 -nlslacZ, to 10 μg of liver DNA extracted from a naïve mouse. Each lane represents an individual mouse. Routes of administration and vector doses are indicated above the lanes. Reprinted with permission of the American Society of Microbiology.