Autologous Ex Vivo Lentiviral Gene Therapy for Adenosine Deaminase Deficiency.

BACKGROUND: Severe combined immunodeficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) is a rare and life-threatening primary immunodeficiency. METHODS: We treated 50 patients with ADA-SCID (30 in the United States and 20 in the United Kingdom) with an investigational gene therapy composed of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with a self-inactivating lentiviral vector encoding human ADA. Data from the two U.S. studies (in which fresh and cryopreserved formulations were used) at 24 months of follow-up were analyzed alongside data from the U.K. study (in which a fresh formulation was used) at 36 months of follow-up. RESULTS: Overall survival was 100% in all studies up to 24 and 36 months. Event-free survival (in the absence of reinitiation of enzyme-replacement therapy or rescue allogeneic hematopoietic stem-cell transplantation) was 97% (U.S. studies) and 100% (U.K. study) at 12 months; 97% and 95%, respectively, at 24 months; and 95% (U.K. study) at 36 months. Engraftment of genetically modified HSPCs persisted in 29 of 30 patients in the U.S. studies and in 19 of 20 patients in the U.K. study. Patients had sustained metabolic detoxification and normalization of ADA activity levels. Immune reconstitution was robust, with 90% of the patients in the U.S. studies and 100% of those in the U.K. study discontinuing immunoglobulin-replacement therapy by 24 months and 36 months, respectively. No evidence of monoclonal expansion, leukoproliferative complications, or emergence of replication-competent lentivirus was noted, and no events of autoimmunity or graft-versus-host disease occurred. Most adverse events were of low grade. CONCLUSIONS: Treatment of ADA-SCID with ex vivo lentiviral HSPC gene therapy resulted in high overall and event-free survival with sustained ADA expression, metabolic correction, and functional immune reconstitution. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01852071, NCT02999984, and NCT01380990.).

Comparison of 2 anal cytology protocols to predict high-grade anal intraepithelial neoplasia.

OBJECTIVES: Nylon-flocked and Dacron swab anal cytology collection procedures were evaluated for detecting high-grade anal intraepithelial neoplasia. MATERIALS AND METHODS: Cross-sectional data for 42 HIV-infected and 16 uninfected men who have sex with men have been used. Sequentially collected anal cytology specimens, high-resolution anoscopy, and medical biopsy evaluated the sensitivity and specificity of cytology for predicting high-grade anal intraepithelial neoplasia. Men showing atypical squamous cells (ASC) or more severe findings by cytology were compared with those showing negative for intraepithelial lesions. RESULTS: The prevalence of high-grade anal intraepithelial neoplasia was 35% (21/58), and findings were approximately 1.5 times higher among HIV-infected compared with uninfected men. Unsatisfactory cytology was twice as common among Dacron compared with nylon-flocked swab protocol specimens (14% [8/58] vs 7% [4/58]). Sensitivity and specificity for the nylon-flocked protocol cytology showing ASC or more severe findings were 81% (58%-95%) and 73% (50%-89%), respectively. Dacron protocol specimens showed 52% (30%-74%) and 58% (34%-80%) sensitivity and specificity, respectively. Men showing ASC or more severe findings using the nylon-flocked protocol cytology showed 3-fold higher odds for high-grade anal intraepithelial neoplasia compared with men with negative results (p < .05), but no statistically significantly higher odds of high-grade anal intraepithelial neoplasia for men showing ASC or more severe findings compared with those with negative results for Dacron protocol cytology (p > .05). CONCLUSIONS: The nylon-flocked protocol better detects high-grade anal intraepithelial neoplasia than does the Dacron protocol, yields more interpretable results, and classifies men with high-grade anal intraepithelial neoplasia as cytologically abnormal 2.5 times more often, even in this small clinical trial. CLINICAL TRIALS REGISTRATION NUMBER: NCT00955591.

Clinical efficacy of gene-modified stem cells in adenosine deaminase-deficient immunodeficiency.

BACKGROUND: Autologous hematopoietic stem cell transplantation (HSCT) of gene-modified cells is an alternative to enzyme replacement therapy (ERT) and allogeneic HSCT that has shown clinical benefit for adenosine deaminase-deficient (ADA-deficient) SCID when combined with reduced intensity conditioning (RIC) and ERT cessation. Clinical safety and therapeutic efficacy were evaluated in a phase II study. METHODS: Ten subjects with confirmed ADA-deficient SCID and no available matched sibling or family donor were enrolled between 2009 and 2012 and received transplantation with autologous hematopoietic CD34+ cells that were modified with the human ADA cDNA (MND-ADA) γ-retroviral vector after conditioning with busulfan (90 mg/m2) and ERT cessation. Subjects were followed from 33 to 84 months at the time of data analysis. Safety of the procedure was assessed by recording the number of adverse events. Efficacy was assessed by measuring engraftment of gene-modified hematopoietic stem/progenitor cells, ADA gene expression, and immune reconstitution. RESULTS: With the exception of the oldest subject (15 years old at enrollment), all subjects remained off ERT with normalized peripheral blood mononuclear cell (PBMC) ADA activity, improved lymphocyte numbers, and normal proliferative responses to mitogens. Three of nine subjects were able to discontinue intravenous immunoglobulin replacement therapy. The MND-ADA vector was persistently detected in PBMCs (vector copy number [VCN] = 0.1-2.6) and granulocytes (VCN = 0.01-0.3) through the most recent visits at the time of this writing. No patient has developed a leukoproliferative disorder or other vector-related clinical complication since transplant. CONCLUSION: These results demonstrate clinical therapeutic efficacy from gene therapy for ADA-deficient SCID, with an excellent clinical safety profile. TRIAL REGISTRATION: ClinicalTrials.gov NCT00794508. FUNDING: Food and Drug Administration Office of Orphan Product Development award, RO1 FD003005; NHLBI awards, PO1 HL73104 and Z01 HG000122; UCLA Clinical and Translational Science Institute awards, UL1RR033176 and UL1TR000124.

Genetic variability of human rotavirus strains isolated from Eastern and Northern India.

An epidemiological study was conducted in Eastern and Northern India to determine the genomic diversity of rotaviruses in these parts of the country. In 2001, a total of 126 Group A rotavirus positive samples were detected from children below 4 years of age with diarrhoea from Kolkata, Dibrugarh and Bhubaneswar in Eastern India, and Chandigarh, a city in Northern India. All the samples were genotyped for VP7 (G-type) and VP4 (P-type) gene by reverse transcription (RT) and multiplex PCR using different type specific primers. The strains with G1P[8] (32.5%) was predominant as reported earlier [Das et al. (2002) J Clin Microbiol 40:146-149] followed by G2P[4](4.7%) and only one sample was of G4P[8] specificity. Along with these common types some rare strains like G1P[6], G2P[8], G2P[6], G4P[4], and G4P[6] were also detected in 14.3% of cases. Thirty percent of samples in this study were mixed infections and 21 (16.7%) specimens remained untypeable either for the VP7 or for the VP4 gene. After sequencing of the VP7 gene, two G9 strains (RMC321 and ISO-3) were identified with P[8] and P[19] specificities. Sequence analysis revealed that they have much lower homology to the G9 strains (116E, INL1, and G16) isolated earlier from Indian subcontinent, but have much higher homology to isolates from Argentina, Brazil, Malawi, Taiwan, and USA suggesting a separate progenitor for these strains.