Impact of Pre-Existing Immunity on Gene Transfer to Nonhuman Primate Liver with Adeno-Associated Virus 8 Vectors

Abstract

Vectors based on the primate-derived adeno-associated virus serotype 8 (AAV8) are being evaluated in preclinical and clinical models. Natural infections with related AAVs activate memory B cells that produce antibodies capable of modulating the efficacy and safety of the vector. We have evaluated the biology of AAV8 gene transfer in macaque liver, with a focus on assessing the impact of pre-existing humoral immunity. Twenty-one macaques with various levels of AAV neutralizing antibody (NAb) were injected intravenously with AAV8 vector expressing green fluorescent protein. Pre-existing antibody titers in excess of 1:10 substantially diminished hepatocyte transduction that, in the absence of NAbs, was highly efficient. Vector-specific NAb diminished liver deposition of genomes and unexpectedly increased genome distribution to the spleen. The majority of animals showed high-level and stable sequestration of vector capsid protein by follicular dendritic cells of splenic germinal centers. These studies illustrate how natural immunity to a virus that is related to a vector can impact the efficacy and potential safety of in vivo gene therapy. We propose to use the in vitro transduction inhibition assay to evaluate research subjects before gene therapy and to preclude from systemic AAV8 trials those that have titers in excess of 1:10.

Liver-directed gene therapy corrects cardiovascular lesions in feline mucopolysaccharidosis type I

“…S1). The expression construct, flanked by AAV2 inverted terminal repeats, was packaged in an AAV serotype 8 capsid and purified on an iodixanol gradient as previously described (33).…”

“…DNA was extracted from tissues and vector genomes quantified by Taqman PCR as previously described (33). …”

Preexisting Neutralizing Antibodies to Adeno-Associated Virus Capsids in Large Animals Other Than Monkeys May Confound In Vivo Gene Therapy Studies

“…AAV8 NAb titer greater than 10 was enough to significantly reduce transduction of the liver. 4 On AAV1-and AAV5-injected animals a NAb titer of 1/160 and 1/80 respectively was enough to significantly inhibit transduction.…”

“…We and others have demonstrated that low levels of preexisting NAb to AAV vectors in monkeys have a profound impact on gene transfer [1][2][3] and redistribution of the vector to other organs such as the spleen. 4 The natural presence of AAV NAbs was not considered to have an impact in nonprimate animal models because their natural AAVs were thought to be serologically different from primate AAVs. A study was conducted to demonstrate this hypothesis.…”

AAV natural infection induces broad cross-neutralizing antibody responses to multiple AAV serotypes in chimpanzees

“…Many species, including dogs, sheep, pigs, macaques, and humans, have serum-circulating neutralizing antibodies (NAbs) to AAV at various levels, [4][5][6][7][8][9][10][11] with a strong correlation between AAV NAbs and the AAV serotype recovered from tissues. For example, the most prevalent AAV serotype in humans, AAV2, is also associated with a higher prevalence of AAV2 NAbs in the population, 10,12,13 whereas, on the contrary, AAV8, a nonhuman AAV, has higher prevalence among rhesus and cynomolgus macaques.…”