Host Responses And Toxicity

Host immune responses, including innate immune responses, cellular immunity, or humoral antibody responses, may hinder the use of some gene therapy vectors. However, administration of AAV vectors has not been reported to induce innate immune responses or proinflammatory cytokines. Also, AAV vectors are replication defective and contain no viral genes, so cellular immune responses against the viral components should not be evoked readily. In all the in vivo studies of AAV vectors in rodents, rabbits, or rhesus macaques, there is little evidence of cellular immune responses to viral components.

AAV vectors may be used mainly for clinical applications requiring only infrequent delivery, but potential humoral immune responses against the viral capsid, either preexisting in the human population or induced by vector administration, must be considered (3,18). Reinfection of humans by AAV is not prevented by serum neutralizing antibodies (3,18). However, more extensive studies will be required to assess whether induction of neutralizing antibody responses will pose any limitations to AAV vectors.

Induction of anti-AAV capsid antibody responses after vector administration may reduce the efficiency of transduc-tion upon readministration (54,196,197). This depends on the route of administration (73,198,199) and also may depend on the quality of the vector preparations. In one study in which 2 AAV vectors expressing the reporter genes bacterial p-ga-lactosidase or human alkaline phosphatase were successively administered to lungs of rabbits, expression from the second administered vector was impaired and this was ascribed to a neutralizing antibody response (200). Similar studies in mice also implied that neutralizing antibodies impaired readministration of AAV vectors, but that this could be partially or completely overcome by transient immunosuppression with anti-CD40 ligand antibodies or soluble CTLA4-immunoglob-ulin at the time of the initial vector administration (197,201). However, interpretation of such studies may be complicated by the expression of the foreign reporter proteins that could represent confounding variables. Furthermore, other studies showed that immune responses were greatly reduced following airway administration of AAV (73) and that vector trans-duction could be seen after at least 3 repeated administrations to the lung of rabbits (32). Thus, up to 3 successive administrations of AAV vectors to rabbit lungs over a 20-week period did not prevent gene expression from the third delivery of vector (32).

It remains to be determined how neutralizing antibody responses to AAV vector capsids might impact applications of AAV vectors. This will most likely require studies in humans to determine if the various animal models such as rodents or rabbits are predictive for the immune response to AAV vectors in humans and whether such immune responses will pose any limitations to their therapeutic application. For relatively infrequent administration of AAV vectors, transient immune blockade (196,197) may not be an attractive option for therapeutic use of AAV or any other gene delivery vectors. An alternate possibility might be to use vectors that have capsids of different serotypes for subsequent administrations (152).

Immune responses to the transgene expressed by an AAV vector vary and may depend on the route of delivery. Both MHC class II-restricted antibody responses and MHC class I cytotoxic T lymphocytes have been reported, but this may vary with the route of administration (199). In some studies, such as intramuscular delivery in mice, there was no immune response to an expressed foreign reporter gene such as bacterial p-galactosidase, and it was suggested that AAV may be a poor adjuvant or may not readily infect professional antigen-presenting cells in muscle (202,203). However, an AAV vector expressing the herpes simplex virus type 2 gB protein was delivered intramuscularly into mice and elicited both MHC class I-restricted CTL responses against the gB protein and anti-gB antibodies (204). Following intramuscular delivery of an AAV human factor IX vector (205,206), there was an antibody response but not a CTL response against the FIX protein. The rules governing immune responses to foreign transgenes following AAV vector delivery remain to be elucidated more fully, but AAV may have utility as a viral vaccination vector (see Section X.H).

There have been few, if any, documented reports or indications of toxicity mediated by AAV vectors. The toxicity of AAV vectors has been extensively tested for a vector expressing the cystic fibrosis transmembrane regulator (CFTR) protein following delivery of these AAV-CFTR vector particles directly to the lung in rabbits and nonhuman primates. In rabbits, the vector persisted and expressed for at least 6 months, but no short- or long-term toxicity was observed and there was no indication of T cell infiltration or inflammatory responses (51). Similarly, in rhesus macaques, AAV-CFTR vector particles were delivered directly to one lobe of a lung and also persisted and expressed for at least 6 months (207). Furthermore, no toxicities were observed by pulmonary function testing, radiological examination, analysis of blood gases and cell counts, and differential in bronchoalveolar lavage or by gross morphological examination or histopathological examination or organ tissues (207). Studies in rhesus macaques were also performed to determine if the AAV-CFTR vector could be shed or mobilized from a treated individual (52). AAV-CFTR particles were delivered to the lower right lobe of the lung, and a high dose of adenovirus and wild-type AAV particles were administered to the nose of the animals. These studies indicated that the vector was not readily mobilized and suggested that the probability of vector shedding and transmission to others is likely to be low. The favorable safety profile of AAV in these preclinical studies has been predictive of a similar safety profile observed in clinical trials of the AAV-CFTR vector in CF patients (9-11). Importantly, preclinical studies of biodistribution of the AAV-CFTR vector following pulmonary delivery in rabbits and macaques showed that there was minimal spread of vector to organs outside the lung, no vector in gonads, and no toxicity was noted in any organ. However, use of different delivery routes may lead to more extensive biodistribution.

An AAV-FIX vector has been studied in preclinical rodent and canine models in support of clinical trials in hemophilia B patients via intramuscular injection or intravenous delivery via the hepatic portal vein. The vector showed a good safety profile when administered by intramuscular injection to hemophilia dogs that was reflected in the intramuscular injection clinical trial (12). However, in a second clinical trial, following intravenous hepatic administration of the AAV-FIX vector, patient semen was positive for the vector genome for a few weeks. This indicated that vector had apparently been distributed to gonads, although further investigation indicated that the vector was not present in sperm and the clinical trial has continued (208). A more extensive study of AAV-FIX vector delivery by both intramuscular and intravenous routes showed that, using a sensitive DNA-PCR assay, there was a dose-dependent detection of vector genomes sequences in the gonads of males of several animal species including mice, rats, rabbits and dogs (208). However, although testis tissue of these species was positive for vector for a short period after delivery, in both rabbits and dogs, semen and sperm were negative for vector sequences, suggesting that the risk of inadvertent germline transmission of vector sequences after intramuscular or intravenous delivery is extremely low (209). Two additional studies of AAV vectors expressing FIX (210) or a-1-antitrypsin (211) genes, after intramuscular delivery into rhesus macaques, again showed excellent safety profiles and did not detect transmission of vector sequences to gonads.

AAV has never been associated with any disease or shown to promote tumorigenesis. Indeed, several studies show that AAV or AAV ITRs can inhibit tumorigenesis (212,213). Also, recent studies in rhesus macaques infected with wild-type AAV2 by intramuscular, intravenous, and intranasal routes showed that, although there were some antibody responses against the AAV capsid, there was no cellular immune response to AAV components and no indication of any tumori-genesis (73). However, there was a recent anecdotal observation of tumors occurring in mice having a homozygous mutation in the p-glucuronidase gene, that were treated as neonates via head vein injection with an AAV vector expressing p-glucuronidase (BGUS). These mice exhibit the lyso-somal storage disease mucopolysaccharidosis VII (Sly syndrome) that is characteristic of the human disease and, if left untreated, the animals die in several months. The mice treated with the AAV-BGUS vector survived up to 18 months and showed a remarkable biochemical and physiological correction of the disease (214). However, at 12 to 18 months some of these mice developed hepatic tumors, but vector genomes could not be found in all the tumors and not at any higher frequency than in nontumor tissue from the same animals (214). Thus, these tumors were probably not caused by inser-tional mutagenesis and clonal expansion. Furthermore, no other studies of AAV vectors, including intravenous delivery in the same animal model and a variety of other animal disease models, have ever shown any tumor formation (214,216). The tumors in these MPSVII mice remains as an anecdotal observation because there were no sham-treated control mice that survived as long. It is possible that the tumor formation is specific for this disease, or the particular animal model, for overproduction of BGUS or to some other unknown cause and does not have any direct relation to the use of an AAV vector. However, some caution needs to be exercised and MPSVII disease possibly may not be an attractive candidate for gene therapy with an AAV vector.

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