Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy

Abstract

Tissues from rhesus monkeys were screened by PCR for the presence of sequences homologous to known adeno-associated virus (AAV) serotypes 1-6. DNA spanning entire rep-cap ORFs from two novel AAVs, called AAV7 and AAV8, were isolated. Sequence comparisons among these and previously described AAVs revealed the greatest divergence in capsid proteins. AAV7 and AAV8 were not neutralized by heterologous antisera raised to the other serotypes. Neutralizing antibodies to AAV7 and AAV8 were rare in human serum and, when present, were low in activity. Vectors formed with capsids from AAV7 and AAV8 were generated by using rep and inverted terminal repeats (ITRs) from AAV2 and were compared with similarly constructed vectors made from capsids of AAV1, AAV2, and AAV5. Murine models of skeletal muscle and liver-directed gene transfer were used to evaluate relative vector performance. AAV7 vectors demonstrated efficiencies of transgene expression in skeletal muscle equivalent to that observed with AAV1, the most efficient known serotype for this application. In liver, transgene expression was 10- to 100-fold higher with AAV8 than observed with other serotypes. This improved efficiency correlated with increased persistence of vector DNA and higher number of transduced hepatocytes. The efficiency of AAV8 vector for liver-directed gene transfer of factor IX was not impacted by preimmunization with the other AAV serotypes. Vectors based on these novel, nonhuman primate AAVs should be considered for human gene therapy because of low reactivity to antibodies directed to human AAVs and because gene transfer efficiency in muscle was similar to that obtained with the best known serotype, whereas, in liver, gene transfer was substantially higher than previously described.

Figure 1

Predicted amino acid sequences for capsid protein VP1 of AAV1, -2, -3A, -3B, -4, -6, -7, and -8. Amino acid sequences of capsid protein VP1 from AAV1 to -4 and AAV6 to -8 were aligned for comparison by using the clustal w program. AAV5 sequence was excluded from the alignment because of its strong divergence from other serotypes. The dots in the alignment represent the amino acids that are identical to those of AAV1 VP1. The dashes indicate the amino acids that are missing at the positions in the alignment.

Figure 2

Expression of a secreted protein A1AT, from AAV vectors injected into murine skeletal muscle and liver. Immune deficient (NCR nudes, A and C) and immune competent mice (C57BL/6, B and D) were treated with different serotypes of AAVCBA1AT (A and B) or AAVAlbA1AT (C and D) through intramuscular or intraportal injections, respectively, at a dose of 1 × 10¹¹ GC per animal. Serum samples were collected retroorbitally at different time points post vector administration and assayed for A1AT concentration. The y axis represents A1AT concentration in the unit of nanogram per milliliter of serum. The x axis represents time points of different bleeds. The data were plotted as average A1AT concentrations of four animals per vector group. The A1AT level in the control animals that received PBS was under the detection limit of the assay.

Figure 3

X-Gal histochemistry of muscle and liver after injection of LacZ-expressing vectors. (Left, Liver) Representative tissue sections from NCR nude mice that received AAV vectors expressing LacZ driven by human thyroid hormone binding globulin gene promoter (TBG), a liver-specific promoter, at a dose of 1 × 10¹¹ GC per animal. The liver tissues were harvested at day 28 post gene transfer for cryosections and X-Gal staining. (Right) Panels show LacZ gene transfer to skeletal muscle of NCR nude mice treated with 1 × 10¹¹ GC of AAVCMVLacZ vectors of different serotypes. The animals were killed at day 10 after vector administration, and injected muscles were harvested for cryosections and X-Gal staining.