TY - JOUR T1 - Age-stratified adeno-associated virus serotype 3 neutralizing and total antibody prevalence in hemophilia A patients from India. JF - J Med Virol Y1 - 2022 A1 - Daniel, Hubert D-J A1 - Kumar, Sanjay A1 - Kannangai, Rajesh A1 - J, Farzana A1 - Joel, Joseph N A1 - Abraham, Aby A1 - Lakshmi, Kavitha M A1 - Agbandje-McKenna, Mavis A1 - Coleman, Kirsten E A1 - Srivastava, Arun A1 - Srivastava, Alok A1 - Abraham, Asha M KW - Adult KW - Animals KW - Antibodies, Neutralizing KW - Antibodies, Viral KW - Child KW - Dependovirus KW - Genetic Vectors KW - Hemophilia A KW - Humans KW - Prevalence KW - Serogroup AB -

Gene therapy using an adeno-associated virus (AAV) vector offers a new treatment option for individuals with monogenetic disorders. The major bottleneck is the presence of pre-existing anti-AAV antibodies, which impacts its use. Even very low titers of neutralizing antibodies (NAb) to capsids from natural AAV infections have been reported to inhibit the transduction of intravenously administered AAV in animal models and are associated with limited efficacy in human trials. Assessing the level of pre-existing NAb is important for determining the primary eligibility of patients for AAV vector-based gene therapy clinical trials. Techniques used to screen AAV-antibodies include AAV capsid enzyme-linked immunosorbent assay (ELISA) and transduction inhibition assay (TIA) for detecting total capsid-binding (TAb) and Nab, respectively. In this study, we screened 521 individuals with hemophilia A from India for TAb and NAb using ELISA and TIA, respectively. The prevalence of TAb and NAb in hemophilia A patients from India were 96% and 77.5%, respectively. There was a significant increase in anti-AAV3 NAb prevalence with age in the hemophilia A patient group from India. There was a trend in anti-AAV3 TAb positivity between the pediatric age group (94.4%) and the adult age group (97.4%).

VL - 94 IS - 9 ER - TY - JOUR T1 - Efficient and error-free correction of sickle mutation in human erythroid cells using prime editor-2. JF - Front Genome Ed Y1 - 2022 A1 - George, Anila A1 - Ravi, Nithin Sam A1 - Prasad, Kirti A1 - Panigrahi, Lokesh A1 - Koikkara, Sanya A1 - Rajendiran, Vignesh A1 - Devaraju, Nivedhitha A1 - Paul, Joshua A1 - Pai, Aswin Anand A1 - Nakamura, Yukio A1 - Kurita, Ryo A1 - Balasubramanian, Poonkuzhali A1 - Thangavel, Saravanabhavan A1 - Marepally, Srujan A1 - Velayudhan, Shaji R A1 - Srivastava, Alok A1 - Mohankumar, Kumarasamypet M AB -

Sickle cell anaemia (SCA) is one of the common autosomal recessive monogenic disorders, caused by a transverse point mutation (GAG > GTG) at the sixth codon of the beta-globin gene, which results in haemolytic anaemia due to the fragile RBCs. Recent progress in genome editing has gained attention for the therapeutic cure for SCA. Direct correction of SCA mutation by homology-directed repair relies on a double-strand break (DSB) at the target site and carries the risk of generating beta-thalassaemic mutations if the editing is not error-free. On the other hand, base editors cannot correct the pathogenic SCA mutation resulting from A > T base transversion. Prime editor (PE), the recently described CRISPR/Cas 9 based gene editing tool that enables precise gene manipulations without DSB and unintended nucleotide changes, is a viable approach for the treatment of SCA. However, the major limitation with the use of prime editing is the lower efficiency especially in human erythroid cell lines and primary cells. To overcome these limitations, we developed a modular lenti-viral based prime editor system and demonstrated its use for the precise modelling of SCA mutation and its subsequent correction in human erythroid cell lines. We achieved highly efficient installation of SCA mutation (up to 72%) and its subsequent correction in human erythroid cells. For the first time, we demonstrated the functional restoration of adult haemoglobin without any unintended nucleotide changes or indel formations using the PE2 system. We also validated that the off-target effects mediated by the PE2 system is very minimal even with very efficient on-target conversion, making it a safe therapeutic option. Taken together, the modular lenti-viral prime editor system developed in this study not only expands the range of cell lines targetable by prime editor but also improves the efficiency considerably, enabling the use of prime editor for myriad molecular, genetic, and translational studies.

VL - 4 ER - TY - JOUR T1 - Erythroid lineage-specific lentiviral RNAi vectors suitable for molecular functional studies and therapeutic applications. JF - Sci Rep Y1 - 2022 A1 - Bagchi, Abhirup A1 - Devaraju, Nivedhitha A1 - Chambayil, Karthik A1 - Rajendiran, Vignesh A1 - Venkatesan, Vigneshwaran A1 - Sayed, Nilofer A1 - Pai, Aswin Anand A1 - Nath, Aneesha A1 - David, Ernest A1 - Nakamura, Yukio A1 - Balasubramanian, Poonkuzhali A1 - Srivastava, Alok A1 - Thangavel, Saravanabhavan A1 - Mohankumar, Kumarasamypet M A1 - Velayudhan, Shaji R KW - Animals KW - Cell Line, Tumor KW - Cell Lineage KW - DNA-Binding Proteins KW - Genetic Vectors KW - Humans KW - Lentivirus KW - Mice KW - RNA Interference KW - RNA, Small Interfering KW - Transcription Factors KW - Transduction, Genetic AB -

Numerous genes exert multifaceted roles in hematopoiesis. Therefore, we generated novel lineage-specific RNA interference (RNAi) lentiviral vectors, H23B-Ery-Lin-shRNA and H234B-Ery-Lin-shRNA, to probe the functions of these genes in erythroid cells without affecting other hematopoietic lineages. The lineage specificity of these vectors was confirmed by transducing multiple hematopoietic cells to express a fluorescent protein. Unlike the previously reported erythroid lineage RNAi vector, our vectors were designed for cloning the short hairpin RNAs (shRNAs) for any gene, and they also provide superior knockdown of the target gene expression with a single shRNA integration per cell. High-level lineage-specific downregulation of BCL11A and ZBTB7A, two well-characterized transcriptional repressors of HBG in adult erythroid cells, was achieved with substantial induction of fetal hemoglobin with a single-copy lentiviral vector integration. Transduction of primary healthy donor CD34 cells with these vectors resulted in >80% reduction in the target protein levels and up to 40% elevation in the γ-chain levels in the differentiated erythroid cells. Xenotransplantation of the human CD34 cells transduced with H23B-Ery-Lin-shBCL11A LV in immunocompromised mice showed ~ 60% reduction in BCL11A protein expression with ~ 40% elevation of γ-chain levels in the erythroid cells derived from the transduced CD34 cells. Overall, the novel erythroid lineage-specific lentiviral RNAi vectors described in this study provide a high-level knockdown of target gene expression in the erythroid cells, making them suitable for their use in gene therapy for hemoglobinopathies. Additionally, the design of these vectors also makes them ideal for high-throughput RNAi screening for studying normal and pathological erythropoiesis.

VL - 12 IS - 1 ER - TY - JOUR T1 - Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin. JF - Elife Y1 - 2022 A1 - Ravi, Nithin Sam A1 - Wienert, Beeke A1 - Wyman, Stacia K A1 - Bell, Henry William A1 - George, Anila A1 - Mahalingam, Gokulnath A1 - Vu, Jonathan T A1 - Prasad, Kirti A1 - Bandlamudi, Bhanu Prasad A1 - Devaraju, Nivedhitha A1 - Rajendiran, Vignesh A1 - Syedbasha, Nazar A1 - Pai, Aswin Anand A1 - Nakamura, Yukio A1 - Kurita, Ryo A1 - Narayanasamy, Muthuraman A1 - Balasubramanian, Poonkuzhali A1 - Thangavel, Saravanabhavan A1 - Marepally, Srujan A1 - Velayudhan, Shaji R A1 - Srivastava, Alok A1 - DeWitt, Mark A A1 - Crossley, Merlin A1 - Corn, Jacob E A1 - Mohankumar, Kumarasamypet M KW - Adenine KW - Anemia, Sickle Cell KW - beta-Globins KW - beta-Thalassemia KW - Cell Line KW - Clustered Regularly Interspaced Short Palindromic Repeats KW - CRISPR-Cas Systems KW - Cytosine KW - Fetal Hemoglobin KW - gamma-Globins KW - Gene Editing KW - Hematopoietic Stem Cells KW - Humans KW - Point Mutation KW - Promoter Regions, Genetic AB -

Naturally occurring point mutations in the promoter switch hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin in sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) and ameliorate the clinical severity. Inspired by this natural phenomenon, we tiled the highly homologous proximal promoters using adenine and cytosine base editors that avoid the generation of large deletions and identified novel regulatory regions including a cluster at the -123 region. Base editing at -123 and -124 bp of promoter induced fetal hemoglobin (HbF) to a higher level than disruption of well-known BCL11A binding site in erythroblasts derived from human CD34+ hematopoietic stem and progenitor cells (HSPC). We further demonstrated in vitro that the introduction of -123T > C and -124T > C HPFH-like mutations drives gamma-globin expression by creating a de novo binding site for KLF1. Overall, our findings shed light on so far unknown regulatory elements within the promoter and identified additional targets for therapeutic upregulation of fetal hemoglobin.

VL - 11 ER - TY - JOUR T1 - Omicron infection increases IgG binding to spike protein of predecessor variants. JF - J Med Virol Y1 - 2022 A1 - Mahalingam, Gokulnath A1 - Periyasami, Yogapriya A1 - Arjunan, Porkizhi A1 - Subaschandrabose, Rajesh Kumar A1 - Mathivanan, Tamil Venthan A1 - Mathew, Roshlin Susan A1 - Ramya Devi, Kt A1 - Premkumar, Prasanna Samuel A1 - Muliyil, Jayaprakash A1 - Srivastava, Alok A1 - Moorthy, Mahesh A1 - Marepally, Srujan AB -

BACKGROUND: SARS-CoV-2 transmission in India in 2020-2022 was driven predominantly by Wild (Wuhan-Hu-1and D614G), Delta, and Omicron variants. The aim of this study was to examine the effect of infections on the humoral immune response and cross-reactivity to spike proteins of Wuhan-Hu-1, Delta, C.1.2., and Omicron.

OBJECTIVES: Residual archival sera (N=81) received between January 2020 and March 2022 were included. Infection status was inferred by a positive SARS-CoV-2 RT-PCR and/or serology (anti-N and anti-S antibodies) and sequencing of contemporaneous samples (N=18) to infer lineage. We estimated the levels and cross-reactivity of infection-induced sera including Wild, Delta, Omicron as well as vaccine breakthrough infections (Delta and Omicron).

RESULTS: We found ~2-fold increase in spike-specific IgG antibody binding in post-Omicron infection compared to the pre-Omicron period, whilst the change in pre- and post-Delta infections were similar. Further investigation of Omicron-specific humoral responses revealed primary Omicron infection as an inducer of cross-reactive antibodies against predecessor variants, in spite of weaker degree of humoral response compared to Wuhan-Hu-1 and Delta infection. Intriguingly, Omicron vaccine-breakthrough infections when compared with primary infections, exhibited increased humoral responses against RBD (7.7-fold) and Trimeric S (Trimeric form of spike protein) (34.6-fold) in addition to increased binding of IgGs towards previously circulating variants (4.2 - 6.5-fold). Despite Delta breakthrough infections showing a higher level of humoral response against RBD (2.9-fold) and Trimeric S (5.7-fold) compared to primary Delta sera, a demonstrably reduced binding (36-49%) was observed to Omicron spike protein.

CONCLUSIONS: Omicron vaccine breakthrough infection results in increased intensity of humoral response and wider breadth of IgG binding to spike proteins of antigenically-distinct, predecessor variants. This article is protected by copyright. All rights reserved.

ER - TY - JOUR T1 - Preferential Expansion of Human CD34CD133CD90 Hematopoietic Stem Cells Enhances Gene-Modified Cell Frequency for Gene Therapy. JF - Hum Gene Ther Y1 - 2022 A1 - Christopher, Abisha Crystal A1 - Venkatesan, Vigneshwaran A1 - Karuppusamy, Karthik V A1 - Srinivasan, Saranya A1 - Babu, Prathibha A1 - Azhagiri, Manoj Kumar K A1 - Chambayil, Karthik A1 - Bagchi, Abhirup A1 - Rajendiran, Vignesh A1 - Ravi, Nithin Sam A1 - Kumar, Sanjay A1 - Marepally, Srujan Kumar A1 - Mohankumar, Kumarasamypet Murugesan A1 - Srivastava, Alok A1 - Velayudhan, Shaji R A1 - Thangavel, Saravanabhavan KW - Animals KW - Antigens, CD34 KW - Fetal Blood KW - Genetic Therapy KW - Hematopoietic Stem Cell Transplantation KW - Hematopoietic Stem Cells KW - Humans KW - Mice KW - Mice, Inbred NOD KW - Mice, SCID AB -

CD34CD133CD90 hematopoietic stem cells (HSCs) are responsible for long-term multilineage hematopoiesis, and the high frequency of gene-modified HSCs is crucial for the success of hematopoietic stem and progenitor cell (HSPC) gene therapy. However, the culture and gene manipulation steps of HSPC graft preparation significantly reduce the frequency of HSCs, thus necessitating large doses of HSPCs and reagents for the manipulation. In this study, we identified a combination of small molecules, Resveratrol, UM729, and SR1 that preferentially expands CD34CD133CD90 HSCs over other subpopulations of adult HSPCs in culture. The preferential expansion enriches the HSCs in culture, enhances the adhesion, and results in a sixfold increase in the long-term engraftment in NSG mice. Further, the culture-enriched HSCs are more responsive to gene modification by lentiviral transduction and gene editing, increasing the frequency of gene-modified HSCs up to 10-fold . The yield of gene-modified HSCs obtained by the culture enrichment is similar to the sort-purification of HSCs and superior to Cyclosporin-H treatment. Our study addresses a critical challenge of low frequency of gene modified HSCs in HSPC graft by developing and demonstrating a facile HSPC culture condition that increases the frequency of gene-modified cells . This strategy will improve the outcome of HSPC gene therapy and also simplify the gene manipulation process.

VL - 33 IS - 3-4 ER -