Rajiv Sharma

All Publications

• ENGINEERING SEQUENTIAL MUTATIONS INTO HUMAN HSPCS YIELDS AN AGGRESSIVE MYELOID MALIGNANCY ALLOWING FOR INTERROGATION OF PRELEUKEMIC TRANSFORMATION Collins, C., Nakauchi, Y., Koehnke, T., Chavez, J., Choi, S., Sharma, R., Zhao, F., Majeti, R. ELSEVIER SCIENCE INC. 2024

  View details for Web of Science ID 001343414100102

• The TRACE-Seq method tracks recombination alleles and identifies clonal reconstitution dynamics of gene targeted human hematopoietic stem cells. Nature communications Sharma, R. n., Dever, D. P., Lee, C. M., Azizi, A. n., Pan, Y. n., Camarena, J. n., Köhnke, T. n., Bao, G. n., Porteus, M. H., Majeti, R. n. 2021; 12 (1): 472

  Abstract

  Targeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may enable the treatment of genetic diseases of the blood and immune system. It is now possible to correct mutations at high frequencies in HSPCs by combining CRISPR/Cas9 with homologous DNA donors. Because of the precision of gene correction, these approaches preclude clonal tracking of gene-targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a methodology that utilizes barcoded AAV6 donor template libraries, carrying in-frame silent mutations or semi-randomized nucleotides outside the coding region, to track the in vivo lineage contribution of gene-targeted HSPC clones. By targeting the HBB gene with an AAV6 donor template library consisting of ~20,000 possible unique exon 1 in-frame silent mutations, we track the hematopoietic reconstitution of HBB targeted myeloid-skewed, lymphoid-skewed, and balanced multi-lineage repopulating human HSPC clones in mice. We anticipate this methodology could potentially be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings.

  View details for DOI 10.1038/s41467-020-20792-y

  View details for PubMedID 33473139

• Induction of Truncating ASXL1 Mutations in Human CD34+HSPCs Mimics Human ASXL1-Mutated Clonal Hematopoiesis and Progression to Myeloid Malignancies Koehnke, T., Sharma, R., Nakauchi, Y., Reinisch, A., Majeti, R. AMER SOC HEMATOLOGY. 2019

  View details for DOI 10.1182/blood-2019-130528

  View details for Web of Science ID 000518218500877

• Barcoded Clonal Tracking of CRISPR-Cas9 and rAAV6-Mediated Gene Targeting in Human Hematopoietic Stem and Progenitor Cells Dever, D. P., Sharma, R., Lee, C. M., Aziz, A., Koehnke, T., Camarena, J., Pan, Y., Zhao, F., Bao, G., Majeti, R., Porteus, M. CELL PRESS. 2019: 5

  View details for Web of Science ID 000464381000008

• Azacitidine and Ascorbate Inhibit the Competitive Outgrowth of Human TET2 Mutant HSPCs in a Xenograft Model of Pre-Leukemia Nakauchi, Y., Thomas, D., Sharma, R., Corces, M., Reinisch, A., Cruz, D., Koehnke, T., Karigane, D., Fan, A., Majeti, R. AMER SOC HEMATOLOGY. 2018

  View details for DOI 10.1182/blood-2018-99-113907

  View details for Web of Science ID 000454837602264

• In vivo genome editing of the albumin locus as a platform for protein replacement therapy BLOOD Sharma, R., Anguela, X. M., Doyon, Y., Wechsler, T., DeKelver, R. C., Sproul, S., Paschon, D. E., Miller, J. C., Davidson, R. J., Shivak, D., Zhou, S., Rieders, J., Gregory, P. D., Holmes, M. C., Rebar, E. J., High, K. A. 2015; 126 (15): 1777-1784

  Abstract

  Site-specific genome editing provides a promising approach for achieving long-term, stable therapeutic gene expression. Genome editing has been successfully applied in a variety of preclinical models, generally focused on targeting the diseased locus itself; however, limited targeting efficiency or insufficient expression from the endogenous promoter may impede the translation of these approaches, particularly if the desired editing event does not confer a selective growth advantage. Here we report a general strategy for liver-directed protein replacement therapies that addresses these issues: zinc finger nuclease (ZFN) -mediated site-specific integration of therapeutic transgenes within the albumin gene. By using adeno-associated viral (AAV) vector delivery in vivo, we achieved long-term expression of human factors VIII and IX (hFVIII and hFIX) in mouse models of hemophilia A and B at therapeutic levels. By using the same targeting reagents in wild-type mice, lysosomal enzymes were expressed that are deficient in Fabry and Gaucher diseases and in Hurler and Hunter syndromes. The establishment of a universal nuclease-based platform for secreted protein production would represent a critical advance in the development of safe, permanent, and functional cures for diverse genetic and nongenetic diseases.

  View details for DOI 10.1182/blood-2014-12-615492

  View details for Web of Science ID 000365451500011

  View details for PubMedID 26297739

  View details for PubMedCentralID PMC4600017

• Robust ZFN-mediated genome editing in adult hemophilic mice BLOOD Anguela, X. M., Sharma, R., Doyon, Y., Miller, J. C., Li, H., Haurigot, V., Rohde, M. E., Wong, S. Y., Davidson, R. J., Zhou, S., Gregory, P. D., Holmes, M. C., High, K. A. 2013; 122 (19): 3283-3287

  Abstract

  Monogenic diseases, including hemophilia, represent ideal targets for genome-editing approaches aimed at correcting a defective gene. Here we report that systemic adeno-associated virus (AAV) vector delivery of zinc finger nucleases (ZFNs) and corrective donor template to the predominantly quiescent livers of adult mice enables production of high levels of human factor IX in a murine model of hemophilia B. Further, we show that off-target cleavage can be substantially reduced while maintaining robust editing by using obligate heterodimeric ZFNs engineered to minimize unwanted cleavage attributable to homodimerization of the ZFNs. These results broaden the therapeutic potential of AAV/ZFN-mediated genome editing in the liver and could expand this strategy to other nonreplicating cell types.

  View details for DOI 10.1182/blood-2013-04-497354

  View details for Web of Science ID 000327466100013

  View details for PubMedID 24085764

  View details for PubMedCentralID PMC3821724