AAT deficiency is caused by mutations in the SERPIN1A gene. The mutation phenotype is designated by its protease inhibitor (PI*) type, with normal AAT designated PI*M. More than 95% of clinical AAT deficiency is due to homozygosity for the PI*Z allele (Glu342Lys) (Crystal et al.
). The range of serum AAT concentrations is 20 to 53
in normal individuals, 3 to 7
in patients with the ZZ phenotype, and 10 to 23
in patients with the SZ phenotype (Brantly et al.
). Epidemiological data indicate that patients with the ZZ phenotype but not the SZ phenotype are at increased risk of developing pulmonary emphysema; achieving trough serum AAT concentrations >11
μg/ml) with weekly intravenous infusions of AAT protein purified from normal plasma donors has been the basis for approval of several products used for augmentation therapy in AAT-deficient patients.
The high serum AAT concentration required to achieve a therapeutic benefit is a major challenge in the development of a gene therapy product for treatment of AAT deficiency. To address this challenge, we have evaluated an rAAV-hAAT vector produced using a recombinant HSV complementation system and an improved column chromatography purification process that achieves much higher yields than previous TFX methods. When evaluated by in vitro assays and compared with TFX-produced vector, the HSV-produced vector had favorable characteristics in terms of purity assessed by silver-stained SDS-PAGE, efficiency of transduction and hAAT expression, and the ratio of full to empty AAV particles in electron micrographs. Compared with the TFX-produced vector, the proportion of empty particles in the HSV-produced vector was lower as assessed by either electron microscopy (2.45% vs. 12.18%) or densitometry of silver-stained bands in SDS-PAGE (24% vs. 34% more VP3 compared with CsCl-purified vector). Both of these methods provide an imprecise estimate of the proportion of empty particles, and neither can fully explain the >100% increase in transgene expression in animals injected with the HSV-produced vector, which suggests that the HSV-produced vector is more infectious or less susceptible to degradation after infection than the TFX-produced vector. Although results in rodents do not necessarily translate to results in humans, if a similar twofold increase in transgene expression levels with the HSV-produced vector compared with the TFX-produced vector were to occur in humans, this might allow for a reduction in the vector dosage required to achieve a therapeutic effect. In addition, the marked increase in vector yield with the HSV-based production method will enable administration of much higher total dosages to patients with AAT deficiency.
Although improved overall yields of rAAV production using the HSV complementation system have been reported previously, it was important to ask if there are any differences in the safety profile of HSV-produced vector compared with TFX-produced vector. Results from the current bridging toxicology study support the safety of rAAV1-CB-hAAT produced using the HSV complementation method when injected IM in mice. There were no local or systemic clinically apparent adverse effects, no adverse effects on hematology or clinical chemistry parameters, and no gross pathology findings. Histological findings at the injection site were minimal to moderate, decreased over time, and their frequency was proportional to hAAT expression as measured by serum hAAT levels. Similar dose-dependent histological changes at the injection site were seen in a previous study in mice injected IM with TFX-produced rAAV1-CB-hAAT (Flotte et al.
At a dose of 1.2
vg/kg, serum hAAT concentrations were higher in mice injected with the HSV-produced vector than in mice injected with the TFX-produced vector. These results are consistent with the higher efficiency of transduction and hAAT expression observed in the in vitro
assays. Higher serum hAAT concentrations were also observed in male mice compared with female mice, especially with the high-dose HSV-produced vector. Sex differences were not observed in a previous toxicology study of rAAV1-CB-hAAT administered by IM injection (Flotte et al.
), but a male sex advantage has been noted after portal vein delivery of rAAV2 and rAAV5 vectors in murine studies of both factor IX and phenylalanine hydroxylase gene transfer (Davidoff et al.
; Mochizuki et al.
). It is possible that the higher serum hAAT levels in male mice in the present study are due to leakage of the vector to the systemic circulation and liver-derived expression due to the use of the CB promoter.
Initial studies with an rAAV vector expressing hAAT packaged in serotype 2 capsid and injected IM in mice indicated that high levels of antibody to hAAT developed in BALB/c mice and appeared to abrogate hAAT expression, whereas immunocompromised nude mice and C57BL/6 mice did not appear to develop antibodies to hAAT, and sustained expression of hAAT was detected in serum from these latter mice (Song et al.
). Subsequent studies in C57BL/6 mice reported that IM injection of an rAAV-hAAT vector packaged in serotype 1 capsid resulted in a marked increase in serum hAAT concentrations compared with the same vector packaged in serotype 2 capsid, and was associated with low but detectable levels of anti-hAAT antibodies (Lu et al.
). In the present study, serum hAAT concentrations tended to decrease with time after dosing, concurrent with development of antibodies to hAAT. Preliminary studies have identified AAT:anti-AAT immune complexes in the serum of C57BL/6 mice injected with rAAV1-CB-hAAT, and dissociation of these immune complexes in vitro
during measurement of serum hAAT concentration by ELISA results in a higher measured AAT concentration (G. Ye, unpublished observations). In contrast, antibodies to hAAT have not been reported among the thousands of patients with AAT deficiency who have received weekly intravenous infusions of purified AAT protein. Thus, results in mice injected with rAAV1-CB-hAAT may underestimate the serum AAT concentrations that will be achieved in patients injected with this vector.
The process used for purification of HSV-produced rAAV1-CB-hAAT is highly effective in removing HSV proteins, and a low-level residual HSV protein (20
ng/ml at the dilution injected) was present in the batch of vector used in the toxicology study. The residual HSV protein is not detectable by immunoblotting (G. Ye, unpublished observations) and is assumed to consist of very small amounts of many different HSV proteins present in HSV-infected cells. This low level of HSV protein appeared to be sufficient to induce low levels of anti-HSV antibodies in mice that received high doses of the HSV-produced vector. It is not known if the lower doses of HSV-produced vector that will be used in clinical trials will result in either an increase in anti-HSV antibodies in the approximately 80% of persons >40 years of age who have preexisting antibodies to HSV (Xu et al.
) or development of anti-HSV antibodies in those who have not been previously infected with HSV. It is unlikely that immune responses to small amounts of HSV protein will have any deleterious effect, because in HSV vaccine studies much larger amounts of HSV proteins combined with strong adjuvants have been administered by IM injection to large numbers of healthy volunteers with no important adverse effects (Corey et al.
; Stanberry et al.
; Bernstein et al.
In summary, results of the preclinical evaluation of rAAV1-CB-hAAT produced using an rHSV complementation system support the planned clinical evaluation of this product.