Class I-restricted T-cell responses to a polymorphic peptide in a gene therapy clinical trial for alpha-1-antitrypsin deficiency

Adeno-associated virus (AAV)-mediated gene therapy is currently being pursued as a treatment for the monogenic disorder alpha-1-antitrypsin (AAT) deficiency. Results from phase I and II studies have shown relatively stable and dose-dependent increases in transgene-derived wild-type AAT after local intramuscular vector administration. In this report we describe the appearance of transgene-specific T-cell responses in two subjects that were part of the phase II trial. The patient with the more robust T-cell response, which was associated with a reduction in transgene expression, was characterized more thoroughly in this study. We learned that the AAT-specific T cells in this patient were cytolytic in phenotype, mapped to a peptide in the endogenous mutant AAT protein that contained a common polymorphism not incorporated into the transgene, and were restricted by a rare HLA class I C alleles present only in this patient. These human studies illustrate the genetic influence of the endogenous gene and HLA haplotype on the outcome of gene therapy

Evaluation of Adeno-Associated Viral Vectors for Liver-Directed Gene Transfer in Dogs

This study evaluated six adeno-associated viral (AAV) vectors expressing green fluorescent protein (GFP) from the liver-specific thyroid hormone–binding globulin (TBG) promoter made with novel capsids in canine liver-directed gene transfer. Studies in 1.5-month-old dogs, which were administered vector through a peripheral vein, showed that AAV8 capsid vectors had the most favorable performance profiles. Interestingly, the absolute levels of hepatocyte transduction achieved with AAV8 were lower in dogs compared with what had been achieved in mice and nonhuman primates. Additional studies were performed with AAV8 delivered into the hepatic artery in adult dogs, with higher doses of vector used to assess potential dose-limiting toxicities. These studies showed good transduction on day 7 in one dog that apparently was lost by day 28 in another dog through the generation of GFP-specific T cells. Each adult dog was carefully monitored for any hemodynamic changes associated with vector infusion. Both animals demonstrated mild to moderate hypotension and bradycardia, which appeared to be anesthesia-related, making it difficult to evaluate contributions of the vector

Results at 2 Years after Gene Therapy for RPE65-Deficient Leber Congenital Amaurosis and Severe Early-Childhood–Onset Retinal Dystrophy

PurposeTo provide an initial assessment of the safety of a recombinant adeno-associated virus vector expressing RPE65 (rAAV2-CB-hRPE65) in adults and children with retinal degeneration caused by RPE65 mutations.DesignNonrandomized, multicenter clinical trial.ParticipantsEight adults and 4 children, 6 to 39 years of age, with Leber congenital amaurosis (LCA) or severe early-childhood–onset retinal degeneration (SECORD).MethodsPatients received a subretinal injection of rAAV2-CB-hRPE65 in the poorer-seeing eye, at either of 2 dose levels, and were followed up for 2 years after treatment.Main Outcome MeasuresThe primary safety measures were ocular and nonocular adverse events. Exploratory efficacy measures included changes in best-corrected visual acuity (BCVA), static perimetry central 30° visual field hill of vision (V30) and total visual field hill of vision (VTOT), kinetic perimetry visual field area, and responses to a quality-of-life questionnaire.ResultsAll patients tolerated subretinal injections and there were no treatment-related serious adverse events. Common adverse events were those associated with the surgical procedure and included subconjunctival hemorrhage in 8 patients and ocular hyperemia in 5 patients. In the treated eye, BCVA increased in 5 patients, V30 increased in 6 patients, VTOT increased in 5 patients, and kinetic visual field area improved in 3 patients. One subject showed a decrease in BCVA and 2 patients showed a decrease in kinetic visual field area.ConclusionsTreatment with rAAV2-CB-hRPE65 was not associated with serious adverse events, and improvement in 1 or more measures of visual function was observed in 9 of 12 patients. The greatest improvements in visual acuity were observed in younger patients with better baseline visual acuity. Evaluation of more patients and a longer duration of follow-up will be needed to determine the rate of uncommon or rare side effects or safety concerns

5 Year Expression and Neutrophil Defect Repair after Gene Therapy in Alpha-1 Antitrypsin Deficiency

Alpha-1 antitrypsin deficiency is a monogenic disorder resulting in emphysema due principally to the unopposed effects of neutrophil elastase. We previously reported achieving plasma wild-type alpha-1 antitrypsin concentrations at 2.5%-3.8% of the purported therapeutic level at 1 year after a single intramuscular administration of recombinant adeno-associated virus serotype 1 alpha-1 antitrypsin vector in alpha-1 antitrypsin deficient patients. We analyzed blood and muscle for alpha-1 antitrypsin expression and immune cell response. We also assayed previously reported markers of neutrophil function known to be altered in alpha-1 antitrypsin deficient patients. Here, we report sustained expression at 2.0%-2.5% of the target level from years 1-5 in these same patients without any additional recombinant adeno-associated virus serotype-1 alpha-1 antitrypsin vector administration. In addition, we observed partial correction of disease-associated neutrophil defects, including neutrophil elastase inhibition, markers of degranulation, and membrane-bound anti-neutrophil antibodies. There was also evidence of an active T regulatory cell response (similar to the 1 year data) and an exhausted cytotoxic T cell response to adeno-associated virus serotype-1 capsid. These findings suggest that muscle-based alpha-1 antitrypsin gene replacement is tolerogenic and that stable levels of M-AAT may exert beneficial neutrophil effects at lower concentrations than previously anticipated

Phase 2 clinical trial of a recombinant adeno-associated viral vector expressing α1-antitrypsin: interim results

Recombinant adeno-associated virus (rAAV) vectors offer promise for the gene therapy of α(1)-antitrypsin (AAT) deficiency. In our prior trial, an rAAV vector expressing human AAT (rAAV1-CB-hAAT) provided sustained, vector-derived AAT expression for \u3e1 year. In the current phase 2 clinical trial, this same vector, produced by a herpes simplex virus complementation method, was administered to nine AAT-deficient individuals by intramuscular injection at doses of 6.0×10(11), 1.9×10(12), and 6.0×10(12) vector genomes/kg (n=3 subjects/dose). Vector-derived expression of normal (M-type) AAT in serum was dose dependent, peaked on day 30, and persisted for at least 90 days. Vector administration was well tolerated, with only mild injection site reactions and no serious adverse events. Serum creatine kinase was transiently elevated on day 30 in five of six subjects in the two higher dose groups and normalized by day 45. As expected, all subjects developed anti-AAV antibodies and interferon-γ enzyme-linked immunospot responses to AAV peptides, and no subjects developed antibodies to AAT. One subject in the mid-dose group developed T cell responses to a single AAT peptide unassociated with any clinical effects. Muscle biopsies obtained on day 90 showed strong immunostaining for AAT and moderate to marked inflammatory cell infiltrates composed primarily of CD3-reactive T lymphocytes that were primarily of the CD8(+) subtype. These results support the feasibility and safety of AAV gene therapy for AAT deficiency, and indicate that serum levels of vector-derived normal human AAT \u3e20 μg/ml can be achieved. However, further improvements in the design or delivery of rAAV-AAT vectors will be required to achieve therapeutic target serum AAT concentrations

Adenoviruses in Lymphocytes of the Human Gastro-Intestinal Tract

Objective: Persistent adenoviral shedding in stools is known to occur past convalescence following acute adenoviral infections. We wished to establish the frequency with which adenoviruses may colonize the gut in normal human subjects. Methods: The presence of adenoviral DNA in intestinal specimens obtained at surgery or autopsy was tested using a nested PCR method. The amplified adenoviral DNA sequences were compared to each other and to known adenoviral species. Lamina propria lymphocytes (LPLs) were isolated from the specimens and the adenoviral copy numbers in the CD4+ and CD8+ fractions were determined by quantitative PCR. Adenoviral gene expression was tested by amplification of adenoviral mRNA. Results: Intestinal tissue from 21 of 58 donors and LPLs from 21 of 24 donors were positive for the presence of adenoviral DNA. The majority of the sequences could be assigned to adenoviral species E, although species B and C sequences were also common. Multiple sequences were often present in the same sample. Forty-one non-identical sequences were identified from 39 different tissue donors. Quantitative PCR for adenoviral DNA in CD4+ and CD8+ fractions of LPLs showed adenoviral DNA to be present in both cell types and ranged from a few hundred to several million copies per million cells on average. Active adenoviral gene expression as evidenced by the presence of adenoviral messenger RNA in intestinal lymphocytes was demonstrated in 9 of the 11 donors tested

Isolation and Characterization of Adenoviruses Persistently Shed from the Gastrointestinal Tract of Non-Human Primates

Adenoviruses are important human pathogens that have been developed as vectors for gene therapies and genetic vaccines. Previous studies indicated that human infections with adenoviruses are self-limiting in immunocompetent hosts with evidence of some persistence in adenoid tissue. We sought to better understand the natural history of adenovirus infections in various non-human primates and discovered that healthy populations of great apes (chimpanzees, bonobos, gorillas, and orangutans) and macaques shed substantial quantities of infectious adenoviruses in stool. Shedding in stools from asymptomatic humans was found to be much less frequent, comparable to frequencies reported before. We purified and fully sequenced 30 novel adenoviruses from apes and 3 novel adenoviruses from macaques. Analyses of the new ape adenovirus sequences (as well as the 4 chimpanzee adenovirus sequences we have previously reported) together with 22 complete adenovirus genomes available from GenBank revealed that (a) the ape adenoviruses could clearly be classified into species corresponding to human adenovirus species B, C, and E, (b) there was evidence for intraspecies recombination between adenoviruses, and (c) the high degree of phylogenetic relatedness of adenoviruses across their various primate hosts provided evidence for cross species transmission events to have occurred in the natural history of B and E viruses. The high degree of asymptomatic shedding of live adenovirus in non-human primates and evidence for zoonotic transmissions warrants caution for primate handling and housing. Furthermore, the presence of persistent and/or latent adenovirus infections in the gut should be considered in the design and interpretation of human and non-human primate studies with adenovirus vectors

Healthcare Engineering Defined: A White Paper

Engineering has been playing an important role in serving and advancing healthcare. The term "Healthcare Engineering" has been used by professional societies, universities, scientific authors, and the healthcare industry for decades. However, the definition of "Healthcare Engineering" remains ambiguous. The purpose of this position paper is to present a definition of Healthcare Engineering as an academic discipline, an area of research, a field of specialty, and a profession. Healthcare Engineering is defined in terms of what it is, who performs it, where it is performed, and how it is performed, including its purpose, scope, topics, synergy, education/training, contributions, and prospects

Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis:a randomised, double-blind, placebo-controlled, phase 2b trial

Background: Lung delivery of plasmid DNA encoding the CFTR gene complexed with a cationic liposome is a potential treatment option for patients with cystic fibrosis. We aimed to assess the efficacy of non-viral CFTR gene therapy in patients with cystic fibrosis. Methods: We did this randomised, double-blind, placebo-controlled, phase 2b trial in two cystic fibrosis centres with patients recruited from 18 sites in the UK. Patients (aged ≥12 years) with a forced expiratory volume in 1 s (FEV1) of 50–90% predicted and any combination of CFTR mutations, were randomly assigned, via a computer-based randomisation system, to receive 5 mL of either nebulised pGM169/GL67A gene–liposome complex or 0·9% saline (placebo) every 28 days (plus or minus 5 days) for 1 year. Randomisation was stratified by % predicted FEV1 (<70 vs ≥70%), age (<18 vs ≥18 years), inclusion in the mechanistic substudy, and dosing site (London or Edinburgh). Participants and investigators were masked to treatment allocation. The primary endpoint was the relative change in % predicted FEV1. The primary analysis was per protocol. This trial is registered with ClinicalTrials.gov, number NCT01621867. Findings: Between June 12, 2012, and June 24, 2013, we randomly assigned 140 patients to receive placebo (n=62) or pGM169/GL67A (n=78), of whom 116 (83%) patients comprised the per-protocol population. We noted a significant, albeit modest, treatment effect in the pGM169/GL67A group versus placebo at 12 months' follow-up (3·7%, 95% CI 0·1–7·3; p=0·046). This outcome was associated with a stabilisation of lung function in the pGM169/GL67A group compared with a decline in the placebo group. We recorded no significant difference in treatment-attributable adverse events between groups. Interpretation: Monthly application of the pGM169/GL67A gene therapy formulation was associated with a significant, albeit modest, benefit in FEV1 compared with placebo at 1 year, indicating a stabilisation of lung function in the treatment group. Further improvements in efficacy and consistency of response to the current formulation are needed before gene therapy is suitable for clinical care; however, our findings should also encourage the rapid introduction of more potent gene transfer vectors into early phase trials