The most significant finding in this study was a clear demonstration of a linear dose–response relationship. This is the first time, to the authors' knowledge, that such a linear relationship between physical dose of a gene therapy product and expression of a therapeutic protein has been shown in humans. Previous clinical trials with the rAAV2-CB-hAAT vector (Brantly et al.
) and with an AAV2 vector expressing clotting factor IX (Manno et al.
) did not have sufficient numbers of subjects with sustained expression at various doses to enable demonstration of a linear dose response, and in clinical trials with an AAV1 vector expressing lipoprotein lipase (Stroes et al.
) the end point was indirect, and thus cannot be compared directly with our results. Our observation indicates that human rAAV gene therapy for AAT deficiency behaves in a predictable fashion and is an important step in the ability to design and conduct appropriate safety and efficacy studies in support of product licensure.
However, AAT is one of the most abundant serum proteins (normal concentration, 20 to 50
or about 1,000 to 2,500
μg/ml), and the peak serum AAT levels achieved after delivery of 6×1012
VG/kg by multiple intramuscular injections (between 412 and 694
, equivalent to 21 to 36
μg/ml) were below the target therapeutic concentration (>11
, equivalent to 572
μg/ml) required to reduce the risk of emphysema. Further improvements in product delivery or product design will therefore be required to achieve therapeutic target serum AAT concentrations. For example, administration of an rAAV1 vector expressing a CTLA4Ig transgene by a regional intravenous method achieved serum concentrations 5- to 8-fold higher than multiple intramuscular injections of the same vector in cynomolgus macaques (Toromanoff et al.
), and suggests that regional vascular delivery of rAAV1-CB-hAAT might result in higher serum AAT concentrations. It is also possible that regional vascular delivery may elevate expression levels by reducing anti-AAV immune responses (Toromanoff et al.
), or that a similar result could be achieved by short-term administration of immunosuppressive drugs. The use of alternative AAV serotypes should also be considered. AAV1 was selected for use in the current clinical trial on the basis of evidence of improved transduction efficiency with AAV1 compared with AAV2 after intramuscular injection in mice (Xiao et al.
; Chao et al.
; Gao et al.
; Rabinowitz et al.
; Lu et al.
). However, more recent data indicate that other serotypes, including recombinant serotypes and other nonnaturally occurring serotypes, may transduce muscle cells more efficiently than AAV1 (Rodino-Klapac et al.
; Asokan et al.
; Qiao et al.
; Pulicherla et al.
). A combination of these approaches may be necessary in order to ultimately achieve the goal of effective gene therapy for AAT deficiency. Results of the current study provide a foundation for designing rational therapeutic protocols.
Results of this study provide additional evidence of the safety of AAV gene therapy. In the highest dose cohort (6×1012
VG/kg), subjects received a total of between 3.3×1014
VG, administered in a total of 135
ml distributed over 100 intramuscular injections, with only mild and transient discomfort at the injection sites.
As expected, all subjects developed anti-AAV antibodies and IFN-γ ELISPOT responses to AAV peptides. In a previous clinical trial with the same vector, anti-AAV immune responses were not associated with any significant decline in peak AAT expression; expression rose irregularly during the first 30 to 180 days and was then sustained at similar levels through day 365. In the present study, serum CK levels were elevated in most subjects in the higher two dose level cohorts on day 30 after injection, which corresponded to the time of peak serum AAT expression, and there was histological evidence of inflammatory cells in muscle biopsy samples 3 months after injections, but no clinical symptoms suggestive of ongoing myositis. It is not known if the T cells seen in muscle biopsies are AAV specific, or if antivector immune responses are responsible for the observed decline in AAT expression after day 30.
We documented T cell response to a single AAT peptide in one subject but found no evidence of untoward clinical effects (no symptoms, no antibody response to AAT, no abnormal liver function tests, and no change in total AAT concentration). The fact that the epitope eliciting this T cell response was distant from the site of the missense mutation is puzzling. Although there is no evidence that the glycosylation pattern of AAT expressed from muscle is different from that of AAT expressed from liver, it is possible that altered glycosylation of AAT may break tolerance and trigger adaptive immune responses in some individuals under certain circumstances. Alternatively, this subject may have had low levels of preexisting reactive T cells that were not detected in the peripheral blood before vector administration. It is reassuring that the three subjects in the highest dose cohort did not mount any detectable T cell responses to AAT peptides.
In summary, results from this clinical trial support the feasibility and safety of AAV gene therapy of AAT deficiency, although further improvements in the design or delivery of rAAV-AAT vectors will be required to achieve therapeutic target serum AAT concentrations.