Gene therapy for alpha-1 antitrypsin deficiency

Abstract

Alpha-1 antitrypsin (AAT) deficiency is a common single-gene disorder among Northern Europeans and North Americans. The carrier frequency for the common missense mutation (Z-AAT) ranges from 4% in the US to nearly 25% in the Republic of Ireland. Severe AAT deficiency (plasma levels below 11 μm) is most commonly associated with an adult-onset lung disease, with pan-acinar emphysema and airway inflammation, which is thought to be primarily owing to the loss of function of AAT in neutralizing neutrophil elastase and other pro-inflammatory enzymes. In 5-10% of patients, severe liver disease may develop. This may occur at any time from infancy to adulthood, and is thought to be owing to toxicity from the Z-AAT mutant protein that folds poorly and forms insoluble polymers within the hepatocyte, which is the primary site for AAT production. Thus, gene therapy for AAT lung disease is conceived of as augmentation of serum levels (a prolonged form of protein replacement, which is currently in use), while gene therapy for liver disease presents the problem of also having to downregulate the production of Z-AAT protein. Over the years, numerous strategies have been employed for the gene therapy of both AAT-deficient lung disease and liver disease. These will be reviewed with an emphasis on modalities that have reached clinical trials recently.

Figure 1.

Concentrations of transgene-derived normal AAT protein in nasal lavage fluid from subjects with PiZZ AAT deficiency. Time-course of responses to intranasal lipoplex delivery of a normal AAT gene with the untreated contralateral nostril serving as control. Data are normalized to total protein concentration. Reprinted with permission from Brigham et al. (9).

Figure 2.

Time-course of vector-mediated AAT expression and enzyme-linked immunosorbent spot (ELISPOT) responses to AAV1 capsid peptides in patients receiving (A) 2.2 × 10¹³ or (B) 6.0 × 10¹³ vgs intramuscularly of a recombinant AAV1 vector expressing the M-AAT gene. Serum M-specific AAT levels are plotted at the top of each panel and interferon-gamma ELISPOT responses to an AAV1 capsid peptide library are plotted at the bottom of each panel. M-AAT levels past day 90 for subjects 201 and 303 are not shown as they resumed protein replacement at this time-point. The day 365 sample for subject 202 could not be tested because of severe hemolysis. ELISPOT responses are characterized as – (negative in both ex vivo and cultured assays),±(positive in cultured assay but negative in ex vivo assay) or + (positive in both ex vivo and cultured assays). Samples for ELISPOT analysis were not available beyond day 90 for subjects 202, 203 and 302. Reprinted with permission from Brantly et al. (15).

Figure 3.

Liver human alpha-1 antitrypsin (hAAT) histology results for PiZ-transgenic mice transduced with AAV8-NC-shRNA or AAV8-3X-shRNA, 14 days post-rAAV8 delivery. Mice transgenic for the human Z-AAT gene were dosed via the portal vein with rAAV8 vectors expressing either three shRNAs directed against the AAT gene (3X-shRNA) or a scrambled negative control (NC-shRNA). (A) Representative hAAT-stained liver sections for mice injected with 1 × 10¹¹ vector genomes of rAAV8-NC-shRNA. (B) Representative hAAT-stained liver sections for mice injected with 1 × 10¹¹ vector genomes of rAAV8-3X-shRNA. (C) Quantification of hAAT-stained areas were performed using MetaMorph Software, sections for each rAAV8-shRNA-transduced livers were analyzed. Thresholded area represents the area on the section that is within the parameters considered to be positively stained. (D) Schematic of the proviral plasmid construct containing three U6 promoter-driven shRNAs, used to make the rAAV8 vectors. Results are expressed as the mean thresholded area for four different livers for each group ± S.E.M. Reprinted with permission from Cruz et al. (20).