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.

https://www.pnas.org/content/pnas/99/18/11854.full.pdf

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

Pre-clinical studies in mice and haemophilic dogs have shown that introduction of an adeno-associated viral (AAV) vector encoding blood coagulation factor IX (FIX) into skeletal muscle results in sustained expression of F.IX at levels sufficient to correct the haemophilic phenotype. On the basis of these data and additional pre-clinical studies demonstrating an absence of vector-related toxicity, we initiated a clinical study of intramuscular injection of an AAV vector expressing human F.IX in adults with severe haemophilia B. The study has a dose-escalation design, and all patients have now been enrolled in the initial dose cohort (2 x 10(11) vg/kg). Assessment in the first three patients of safety and gene transfer and expression show no evidence of germline transmission of vector sequences or formation of inhibitory antibodies against F.IX. We found that the vector sequences are present in muscle by PCR and Southern-blot analyses of muscle biopsies and we demonstrated expression of F.IX by immunohistochemistry. We observed modest changes in clinical endpoints including circulating levels of F.IX and frequency of FIX protein infusion. The evidence of gene expression at low doses of vector suggests that dose calculations based on animal data may have overestimated the amount of vector required to achieve therapeutic levels in humans, and that the approach offers the possibility of converting severe haemophilia B to a milder form of the disease.

Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector

Adeno-associated virus (AAV) is a human virus that can be propagated either as an integrated provirus or by lytic infection (Atchison et al. 1965; Hoggan et al. 1966, 1972). The ability to form a latent infection appears to be an integral part of the AAV life cycle. Except under special circumstances (Yacobson et al. 1987; Schlehofer et al. 1986; Yalkinoglu et al. 1988), AAV requires the presence of a helper virus to initiate a productive viral infection (Fig. 1). Members of either the herpes or adenovirus families can provide the necessary helper functions (Atchison et al. 1965; Melnick et al. 1965; Hoggan et al. 1966; Buller et al. 1981; McPherson et al. 1985) and vaccinia virus can provide at least partial helper function (Schlehofer et al. 1986). In the absence of a helper virus AAV produces no progeny virus, but instead integrates into a host chromosome (Hoggan et al. 1972; Berns et al. 1975; Handa et al. 1977; Cheung et al. 1980). With rare exceptions, AAV proviruses appear to be stable. However, if a cell line that is carrying an AAV provirus (Fig. 1) is subsequently superinfected with a helper virus, the AAV genome is excised and proceeds through a normal productive infection (Hoggan et al. 1972; Cheung et al. 1980). This ability to establish a latent infection which can later be rescued appears to be a mechanism for ensuring the survival of AAV in the absence of a helper virus. The unusual life cycle of AAV has led a number of parvovirus laboratories to explore the possibility of using AAV as a general mammalian transduction vector.

Use of adeno-associated virus as a general transduction vector for mammalian cells.

https://diyhpl.us/~bryan/papers2/gene-therapy/Adeno-associated%20virus%20serotypes%20-%20vector%20toolkit%20for%20human%20gene%20therapy%20-%202006.pdf

Adeno-associated virus serotypes: vector toolkit for human gene therapy.

Recombinant adeno-associated viruses (AAVs) have unique gene-transfer properties that speak to their potential as carriers for gene therapy or vaccine applications. However, the presence of neutralizing antibodies to AAV as a result of previous exposure can significantly limit effective gene transfer. In this study, we obtained 888 human serum samples from healthy volunteers in 10 countries around the world. Samples were assayed for neutralizing antibodies to AAV1, AAV2, AAV7, and AAV8, as well as to a novel, structurally distinct AAV vector, rh32.33, in an in vitro transduction inhibition assay. Our data revealed that neutralizing antibodies to AAV2 were the most prevalent antibodies in all regions, followed by antibodies to AAV1. The seroprevalences of antibodies to AAV7 and to AAV8 were lower than that for antibodies to AAV1, and neutralization of AAVrh32.33 was only rarely detected. Our data also indicate a strong linkage of seroreactivity between apparently distinct serotypes that has not been predicted previously in animal models.

Worldwide Epidemiology of Neutralizing Antibodies to Adeno-Associated Viruses

The seroepidemiology of the defective adenovirus-associated satellite viruses was studied by use of a neutralization test that has been shown to be much more sensitive than the complement-fixation (CF) test. Less than 25% of the subjects in any age group were positive for any type of satellite virus CF antibody. The neutralization test revealed serological evidence of satellite infection in almost 80% of the subjects in some age groups. Studies of geometric mean antibody titers by neutralization showed that antibody against type 1 and the type 2-3 complex reached peak levels at 2 to 3 years of age; antibody against types 2 and 3 reached mean titers of 500 to 800, whereas mean antibody levels of only 200 were found for type 1. Type 4 antibodies were rarely noted in human sera. In a companion study in monkeys, monospecific responses to types 1 and 4 were found only in rhesus and green monkey sera, respectively. These data suggest that the type 2-3 complex may be the only naturally occurring satellite serotype which infects man and that antibodies noted against types 1 and 4 may be the result of cross-reactions. No satellite viruses were isolated from throat or rectal swabs of 100 children between 6 months and 3 years of age. However, the patterns of adenovirus and enterovirus isolations from these specimens were typical of previous studies in this laboratory.

Seroepidemiological and Ecological Studies of the Adenovirus-Associated Satellite Viruses

In a study being reported concurrently, we confirmed the observation of Deinhardt et al. [1] that a pool of fifth passage sera from marmosets originally inoculated with human hepatitis material was transmissible in marmosets and produced a hepatitislike picture [2]. That study characterized the infection in marmosets following inoculation with the Barker strain of marmoset hepatitis virus and reported some physical characteristics of the agent. Histological studies demonstrated in the livers of infected animals a characteristic inflam-

Attempted Isolation of Hepatitis Viruses in Marmosets

The study of human viral hepatitis has been hampered by lack of susceptible tissue cultures or animals [1, 2]. An encouraging lead was recently obtained by Deinhardt et al. [3] who inoculated human sera into marmosets and obtained from the serum of certain inoculated animals a transmissible agent that produced histologic hepatitis associated with a rise in serum transaminase. Their initial report dealt with the isolation of the virus from human materials and with the clinical and histological features of the infection in marmosets. Hepatitis was not found in control animals in their study. A brief report [4] noted that commercial pooled human IgG strikingly reduced the infectivity of infectious marmoset sera, but this could not be confirmed in later experiments [5]. Using infectious marmoset sera kindly supplied by Deinhardt and Holmes, we have confirmed the existence of a transmissible hepatitis in marmosets and have investigated the physical properties of the virus and the characteristic features of the infection.

https://www.jstor.org/stable/info/30102204

Wade P. Parks

Papers

Seroepidemiological and Ecological Studies of the Adenovirus-Associated Satellite Viruses

Attempted Isolation of Hepatitis Viruses in Marmosets

Characterization of Marmoset Hepatitis Virus

Failure of a replicating adenovirus to enhance adeno-associated satellite virus replication

Seroepidemiological andEcological Studies ofthe Adenovirus-Associated