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943 Single-Cell-Multiomics Demonstrates Molecular Efficacy of a Clinical Lentiviral Vector for Gene Therapy of RPS19-Deficient Diamond-Blackfan Anemia

Program: Oral and Poster Abstracts
Type: Oral
Session: 509. Bone Marrow Failure and Cancer Predisposition Syndromes: Congenital and Misc.
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Biological therapies, Translational Research, Bone Marrow Failure Syndromes, Inherited Marrow Failure Syndromes, Diseases, Gene Therapy, Therapies, Biological Processes, Technology and Procedures, pathogenesis, omics technologies
Monday, December 11, 2023: 4:30 PM

Sofia Freiman, PhD1*, Jun Chen, MD, PhD2*, Johan Flygare, MD, PhD1, Chayenne Kampman3*, Karin Pike-Overzet3*, Alexander Mohseny, MD, PhD3*, Teng-Cheong Ha, PhD4*, Mathias Lidgren, MD PhD1*, Shamit Soneji5*, Stefan Karlsson, MD6*, Dagmar Pospíšilová, MD, PhD7*, Frank Staal8*, Axel Schambach, MD, PhD9* and Johan Flygare, MD, PhD1*

1Lund Stem Cell Center, Lund University, Lund, Sweden
2Lund Srtem Cell Center, Lund University, Lund, Sweden
3Leiden University Medical Center, Leiden, NLD
4Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
5Lund Stem Cell Centre, Lund University, Lund, Sweden
6Lund University, Lund, SWE
7Department of Pediatrics, Palacky University and University Hospital, Olomouc, Czech Republic
8Leiden University Medical Center, Leiden, Netherlands
9Institute of Experimental Hematology, Hannover Medical School, Hannover, DEU

It has previously been demonstrated that lentiviral vector-mediated gene transfer of human codon-optimized RPS19 to hematopoietic stem cells by the gene therapy vector (CLIN-LV-EFS-coRPS19-PRE*) corrects the anemic phenotype of RPS19-deficient mice, supporting development of this vector for clinical gene therapy of RPS19-deficient Diamond-Blackfan Anemia (DBA). In this study, we evaluate the molecular efficacy of CLIN-LV-EFS-coRPS19-PRE* in CD34+ cells from RPS19-deficient DBA patients.

CD34+ cells from two healthy donors and three DBA patients with confirmed heterozygous mutations in RPS19 were transduced using GMP-like reagents according to a protocol developed for clinical use. To evaluate the therapeutic effect of RPS19-gene transfer, transduced (GT) and untransduced (Mock) CD34+ cells were cultured under conditions supporting erythroid and myeloid progenitor proliferation and differentiation. In order to obtain a comprehensive molecular characterization of the therapeutic mechanisms, cells from day 9 of culture were subjected to CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) analysis, a single-cell-multiomics method combining the advantages of FACS and scRNA-Seq for simultaneously analyzing transcriptomes alongside cell surface protein abundance at the single cell level. In GT cells, transduced (coRPS19-positive) cells were identified based on transgene expression. At day 9, 84-91% of erythroid progenitors in the GT-DBA samples expressed the coRPS19-transgene, while the frequency in myeloid progenitors was 29-56%. To reveal the molecular therapeutic effect of GT, we compared coRPS19-positive cells to Mock-treated cells from the same individuals. Expression of coRPS19 in DBA erythroid progenitor cells led to a significant induction of genes associated with terminal erythropoiesis (HEMGN, HBB, AHSP, EPB42 and GYPA) and down-regulation of genes associated with apoptosis and p53 activation (BAX, MDM2, ZMAT3 and MIR34AHG). GT also induced up-regulation of the large non-coding RNA LINC01133 and down-regulation of XACT. Interestingly, the two most-significantly changed genes in coRPS19-positive erythroid cells in all DBA samples were RPL22L1 and CD70. RPL22L1 is an RNA-binding component of the 60S ribosomal subunit that regulates pre-mRNA splicing but is not required for global cap-dependent translation. CD70 mRNA and protein exclusively expressed in erythroid progenitors in Mock-treated DBA samples and down-regulated in coRPS19-positive cells. CD70 is thus a new potential marker of DBA erythroid progenitor cells with a possible role in DBA pathogenesis. Myeloid progenitor DBA cells primarily responded to GT by up-regulation of ribosomal protein genes, suggesting RPS19-deficiency in myeloid progenitors leads to reduced ribosome biogenesis without the nucleolar stress observed in erythroid cells.

To summarize, GT-induced changes in gene and protein expression agree with restoration of healthy ribosome biogenesis and elimination of nucleolar stress-induced p53 activation in erythroid progenitor cells responsible for the DBA phenotype, demonstrating molecular efficacy of CLIN-LV-EFS-coRPS19-PRE* supporting development for clinical gene therapy. In addition, the GT-induced reversal of RPS19-deficiency reveals several genes with potential relevance in DBA diagnostics and pathogenesis, such as RPL22L1 and CD70 for further investigation.

Disclosures: Karlsson: Apriligen: Consultancy. Flygare: Apriligen: Consultancy.

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*signifies non-member of ASH