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9.2.1 General Toxicity and Inflammation

The risks of participating in this study cannot be predicted, since this is the first time that AMT-010 is administered to human subjects. However, pre-clinical studies with AMT-010 (section 1.8), and pre-clinical and clinical studies with other AAV vectors indicate that the risk of side effects is very low. A previous clinical study using an AAV2-derived vector expressing factor IX in hemophilia B patients has indeed shown that intramuscular injection of an AAV vector at multiple sites was well tolerated with no significant systemic or local toxicities in 8 human subjects (Manno et al., 2003). Doses used in this study ranged from 2x1011 to 1.8x1012 gc/kg, which is higher than the doses proposed in this study.

Toxicity studies in mice with AMT-010 showed no effect on general well-being of the mice with doses up to 1x1013 gc/kg. In addition, no abnormalities in blood clinical chemistry and haematological parameters were observed. The only dose-related clinical observation was a reduction in gain in body weight in the highest virus dose group. This reduction did not correlate with a decrease in food intake. LPL is the key enzyme in lipid metabolism, and it is possible that the elevated LPL levels in the high dose group influence general metabolism.

Histopathological investigations of mice injected with 1x1013 gc/kg AMT-010 did not reveal gross signs of toxicity. There were no macroscopic findings. We did observe signs of both short-and long-term inflammatory responses to AMT-010. There was a transient increase in serum amyloid A levels (dissipating within 24 hours), indicating an acute inflammatory response. There was a minimal and reversible lymphoid hyperplasia in the spleen (day 8 and 29). This transient lymphoid hyperplasia is probably related to lymphocyte activation triggered by the viral capsid proteins, following vector administration. Such an immune response to the viral capsids has been detected in all pre-clinical animal studies. A mild (grade 1) myositis was observed in all administration groups (day 8), consistent with needle track lesions expected after intramuscular injection. At day 91, a minimal (grade 1) to slight (grade 2) myositis was observed at the injection sites of animals treated with 1x1012 and 1x1013 _{gc/kg AMT-010, respectively, whereas this was not observed in the control and}

1x1011 gc/kg group.

In summary, both short- and long-term inflammatory events were observed in rodents at a dose that exceeds the starting dose in humans 10- to 100-fold. These observations have prompted special focus on administration related toxicity within the injected muscle. Measures to document such developments have been included in this study (see sections 6.1 and 6.3). It should be noted, however, that pre-clinical studies in LPL-deficient mice have shown that administration of AMT-010 actually improved muscle tissue health. Muscle

morphology is abnormal in these mice due to the disturbed lipid metabolism, and was improved by administration of AMT-010.

9.2.2 Vector Dissemination to other Tissues and Germline Transmission

Biodistribution studies in mice showed vector dissemination to filtering organs (liver, spleen, marrow) shortly after intramuscular administration. These levels dissipated rapidly in the ensuing weeks following administration. Apart from the injected muscle, persistence of AMT-010 vector DNA was also observed in the draining lymph nodes, close to the injection site. Analysis of mice 3 months after intramuscular injection of the proposed starting clinical dose (1x1011 _{gc/kg) showed that significant levels of AMT-010 vector DNA could be}

detected only in injected muscles, and in inguinal lymph nodes. Very low amounts of AMT- 010 vector DNA were detected at this time point in testes (2 out of 5 negative; remainder <10 copies/µg genomic DNA), epididymis (4 out of 5 negative; remainder <10 copies/µg genomic DNA), and ovaries (5 out of 5 negative). At higher doses (1x1013 gc/kg), AMT-010 vector DNA was detected in all gonads after 90 days, but absolute levels were low. Additional biodistribution data from LPL deficient cats further confirmed that dissemination of AMT-010 vector to gonads is very limited at dosages up to 1.7x1012 gc/kg (<10-170 copies/µg genomic DNA). As one microgram of genomic DNA represents approximately 300,000 haploid mammalian genomes, the probability of germline transmission of vector sequence is between <1:30,000 and 1:600. Since only a very low proportion of these vector sequences integrate (see section 9.2.3), it is evident that the rate of insertional mutation by AMT-010 is much lower than the rate of endogenous insertion by L1 retrotransposons, which is 1 in every 50-100 human sperm (Kazazian, 1999) and lower than the 90% probability that a human sperm carries a spontaneous amino-acid altering mutation (Department of Health and Human services, National Institutes of Health Recombinant DNA Advisory committee, Minutes of meeting. National Institutes of Health, Bethesda, MD, March 11-12 2000; Human gene therapy 11, 1231-1252). Importantly, although AMT-010 vector DNA can be detected in gonadal tissue, this does not prove that germ line cells are transduced by the vector. Oöcytes are relatively inaccessible, because the vector needs to traverse the thecal layer, follicle wall, layer of follicle cells and the zona pellucida. To efficiently transduce the spermatogonia, the vector must traverse the basement membranes of two structures, the vascular wall and the seminiferous tubule. This is in line with the fact that hardly any vector DNA (0-<10 copies/sample) was detected in motile sperm of cats injected intramuscularly with AMT-010 at dosages up to 1.7x1012 gc/kg (see section 1.8.2). Previous studies with AAV2 vectors also have shown that vector DNA is highly unlikely to end up in germ line cells. For instance, intramuscular administration of AAV2 did result in vector dissemination to the testes, but no signal could be detected in sperm cells (Arruda et al., 2001). Vector DNA was located extracellulary in the testis basement membrane, and interstitial space. Exposure of mouse spermatozoa to very high concentrations of vector failed to lead to germ cell transduction (Couto et al., 2004). Furthermore, application of the same vector in a Phase I trial also failed to show presence of vector DNA in sperm cells of male subjects (Manno et

In summary (see also 1.8.2), there is vector leakage from the injected muscle shortly after administration of AMT-010, via the circulation to filtering organs (liver, spleen, marrow), and via lymph drainage to inguinal lymph nodes. Levels of AMT-010 vector DNA do not persist in the filtering organs, but may contribute to initial inflammatory responses (similar to persistence in lymph nodes). It should be mentioned that transgene expression has not been observed in the liver after intramuscular delivery of AMT-010 to mice and cats, and therefore dissemination of vector DNA to this organ does not correlate with effective transduction.Very low levels of AMT-010 vector DNA were detected in gonadal tissue. These levels decreased over time, and the very low to undetectable levels in motile sperm suggest that germ line transmission of AMT-010 DNA is unlikely.

9.2.3 Integration

It is very unlikely that the study agent could damage the DNA in the cell of the subject’s muscle, putting the subject at risk for developing cancer in the future. The majority of AAV vector DNA appears to persist as episomal rather than integrated DNA, which makes it very unlikely that mutagenesis will occur. An extensive study on the genetic fate of AAV vector genomes in mice muscle demonstrated that >99.5% persists primarily as large and small episomal concatemers (Schnepp et al., 2003a). Similar results were obtained after intramuscular delivery of AAV vector to rabbits (Schnepp et al., 2003b). Based on these studies, the integration frequency of AAV vectors is in the same order of magnitude as plasmid DNA, and 200-250 fold lower than the spontaneous mutation rate of 10-5/gene. This is in line with a recent study by Miller et al. (2004), suggesting that AAV vectors lack the machinery for specific integration and integrate at existing chromosome breaks, rather than causing breaks. The integration frequency of AAV vectors in liver tissue has been less well quantified, but studies also support that the majority of vector is present as episomal concatamers (Nakai et al., 2002). Altogether, these observations indicate that the risk for inadvertent integration events such as the development of leukaemia recently observed in SCID children due to integration and activation of an oncogene by a retroviral vector is much lower in case of AAV vectors. Intramuscular delivery of AMT-010 in mice resulted in low transient levels of vector DNA in dividing marrow cells at a dose of 1x1011 _{gc/kg (90-930}

copies/µg genomic DNA at day 8, decreasing to 0 copies/µg genomic DNA at day 91). Vector DNA in marrow also diminished rapidly over time, after injection of 1x1013 gc/kg (section 1.8.2), indicating that there is no persistence of vector sequence and that therefore the likelihood of integration, even in dividing tissue, is low. Furthermore, efforts to transduce human hematopoietic stem cells using AAV vectors were often not successful and these poor results may well be indicative of the low risk for inadvertent transduction of these cells (Srivastava, 2004).

Although it remains difficult to be certain whether there is a risk of tumor formation, our pre- clinical data support that this risk is minimal. A substantial number of mice and cats have been injected with AMT-010 and some animals were followed for more than 1-1.5 years. To date, the development of cancer has not been observed and no evidence has been obtained that particular sequences of AMT-010 support integration and/or carcinogenesis. Furthermore, a large number of animal studies were performed using different AAV vectors,

and approximately 120 human subjects have been injected with AAV vectors without development of cancer. The exception is in one study using AAV-ß glucuronidase in newborn mice (Daly et al., 1999). Liver tumors were reported, but there has not been any evidence to indicate that these tumors were caused by DNA damage from the vector, since vector DNA was not present in most of the tumors sampled.

9.2.4 Overexpression of LPL

Over-expression of LPL has been reported to cause muscle myopathy (Levak-Frank et al., 1995; Hoefler et al., 1997) and insulin resistance (Kim et al., 2001). These effects were observed in transgenic mice with 5 to 24-fold muscle-specific over-expression of LPL. Deleterious effects were not observed in transgenic rabbits, containing 80-fold higher than normal levels of human LPL (Koike et al., 2002). Whole-body or muscle-specific insulin resistance upon over-expression of LPL was not found by Voshol and co-workers (Voshol, et al., 2001). Administration of AMT-010 resulted in over-expression in LPL-/- mice by 11 to 24-fold, depending on dose/site, without detecting any adverse effects. In contrast, untreated LPL-/- mice displayed disturbed muscle morphology, and administration of AMT-010 resulted in improvement of this disturbed muscle morphology. Furthermore, in transgenic LPL over-expressing mice, myopathy was attributed to an increased flux of free fatty acids (FFA) into the tissue. Untreated LPL-/- mice displayed increased muscle lipid levels (FFA, TG, and Tchol) and these levels were normalised upon administration of AMT-010.

In summary, some studies indicate that local over-expression of LPL may cause muscle toxicity. Our own studies in LPL-/- mice show beneficial effects of AMT-010 on muscle morphology. Measures to monitor muscle function and signs of possible local muscle toxicity have been included in this protocol, to address these issues.

9.2.5 Immune Responses

Administration of AMT-010 results in the generation of anti-AAV antibodies in animal models, and will likely do so in human subjects. This will not affect initial transduction of muscle tissue, as AAV infects the cells before the onset of anti-AAV antibodies, but may have an effect on the duration of expression. It is an issue for re-administration of a similar vector in the same individual, but this is not part of the current study. In any case, adverse effects or muscle-specific toxicity related to the generation of antibodies against AAV1 were not observed.

It is possible that antibodies against LPLS447X, the transgene product, will develop in humans. This is intuitively more likely in those who have homozygous null mutations who have never produced LPL, active or otherwise. In LPL-/- cats, administration of AMT-010 results in the generation of anti-LPL (inhibitory) antibodies. CTL responses have not been assessed, but the transient increase in CPK levels concurrent with a loss of efficacy and transgene expression may indicate damage and destruction of transduced muscle tissue. As stated, LPL-/- mice treated with AMT-010 do not develop anti-LPL antibodies. The likelihood that subjects in the present study develop antibodies to LPL will be minimised by only including subjects that do have LPL mass levels >5% of normal. However, immune responses to the LPLS447X protein

may still occur, due to differences in conformation between the LPLS447X protein and the endogenous inactive LPL protein.