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CD34+CD133+CD90+ hematopoietic stem cells (HSCs) are responsible for long-term multi-lineage 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 ex vivo 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. Here, we identified a combination of small molecules, Resveratrol, UM729, and SR1 that preferentially expands CD34+CD133+CD90+ HSCs over other subpopulations of adult HSPCs in ex vivo culture. The preferential expansion enriches the HSCs in ex vivo culture, enhances the adhesion and results in a 6-fold 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 in vivo. 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 in vivo. This strategy will improve the outcome of HSPC gene therapy and also simplify the gene manipulation process.

β-thalassemia and HbE result from mutations in the β-globin locus that impedes the production of functional β-hemoglobin and represents one of the most common genetic disorders worldwide. Recent advances in genome engineering have opened up new therapeutic opportunities to directly correct these pathogenic mutations using base editors that install transition mutations (A>G and C>T) in the target region with minimal generation of indels. Herein, for the first time, we demonstrate the usage of base editor in the correction of point mutations spanning multiple regions of the HBB gene, including promoter, intron and exon. To this end, we have engineered human erythroid cells harbouring the diverse HBB mutations, thus eliminating the requirement of patient CD34+ HSPCs with desired mutations for the primary screening by base editors. We further performed precise creation and correction of individual HBB point mutations in human erythroid cells using base editors, which were effectively corrected in the HBB-engineered erythroid model. Intriguingly, most bystander effects produced by the base editor at the target site were reported to exhibit normal hemoglobin variants. Overall, our study provides the proof-of-concept for the precise, efficient and scarless creation and correction of various pathogenic mutations at the coding and non-coding regions of HBB gene in human erythroid cells using base editors and establishes a novel therapeutic platform for the treatment of β-thalassemia/HbE patients. This study can be further explored in correcting the other monogenic disorders caused due to single base substitutions.

Ex vivo genetic manipulation of autologous hematopoietic stem and progenitor cells (HSPCs) is a viable strategy for the treatment of hematologic and primary immune disorders. Targeted genome editing of HSPCs using the CRISPR-Cas9 system provides an effective platform to edit the desired genomic locus for therapeutic purposes with minimal off-target effects. In this chapter, we describe the detailed methodology for the CRISPR-Cas9 mediated gene knockout, deletion, addition, and correction in human HSPCs by viral and nonviral approaches. We also present a comprehensive protocol for the analysis of genome modified HSPCs toward the erythroid and megakaryocyte lineage in vitro and the long-term multilineage reconstitution capacity in the recently developed NBSGW mouse model that supports human erythropoiesis.

CD34

CRISPR/Cas9 system, a bacterial adaptive immune system developed into a genome editing technology, has emerged as a powerful tool revolutionising genome engineering in all branches of biological science including agriculture, research and medicine. Rapid evolution of CRISPR/Cas9 system from the generation of double strand breaks to more advanced applications on gene regulation has made the wide-spread use of this technology possible. Medical science has benefited greatly from CRISPR/Cas9; being both a versatile and economical tool, it has brought gene therapy closer to reality. In this review, the development of CRISPR/Cas9 system, variants thereof and its application in different walks of medical science- research, diagnostics and therapy, will be discussed.

Switching hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin is an effective strategy for the treatment of beta-hemoglobinopathies. Fetal hemoglobin expression is down-regulated in the postnatal period due to the interplay of transcription regulators with the HBG promoters. However, in the hereditary persistence of fetal hemoglobin (HPFH) condition, naturally occurring point mutations in the HBG promoter causes continued expression of fetal globin even during adulthood. Inspired by this natural phenomenon, we screened the proximal promoter of human HBG genes using adenine and cytosine base editors to identify other nucleotide substitutions that could potentially lead to elevated levels of fetal globin. Both the base editors efficiently and precisely edited at the target sites with a minimal generation of indels and no deletion of one of the duplicated HBG genes. Through systematic tiling across the HBG proximal promoter, we identified multiple novel target sites that resulted in a significant increase in fetal globin levels. Further, we individually validated the top eight potential target sites from both the base editors and observed robust elevation in the fetal globin levels up to 47 %, without any detrimental effects on erythroid differentiation. Our screening strategy resulted in the identification of multiple novel point mutations and also validated the known non-deletional HPFH mutations that could elevate the fetal globin expression at therapeutically relevant levels. Overall, our findings shed light on so far unknown regulatory elements within the HBG promoter that normally mediates fetal globin silencing and identify additional targets for therapeutic upregulation of fetal hemoglobin.

Purpose. This study aimed to characterize A. baumannii strains isolated from patients in an intensive care unit (ICU) setting. Molecular techniques were used to study clonal relatedness and determine a fast, efficient and cost-effective way of detecting persistent clones. Methodology. A. baumannii (n=17) were obtained in June and November 2015 from a single ICU setting in South India. DNA typing methods such as multilocus sequence typing (MLST), single-locus sequence-based typing (SBT) and DNA fingerprinting PCRs (M13, DAF4 and ERIC2) were employed to understand the association of clones. PCRs were performed for the antimicrobial resistance genes ISAba1-bla OXA-51-like, ISAba1-bla OXA-23-like, bla NDM-1, bla PER-7 and bla TEM-1, and the virulence genes cpa 1, cpa2 and pkf. Results. The MLST showed some degree of corroboration with the other DNA typing methods. The M13 PCR was found to give better results than the other fingerprinting methods. ST848 (CC92) was the dominant strain isolated in both June and November. All isolates were bla OXA-51-like-positive, with 16 having ISAba1 upstream of the bla OXA-51-like and bla OXA-23-like genes. Genes such as bla NDM-1 (23 %, n=4), bla PER-7 (58.8 %, n=10), pkf (82 %, n=14), bla TEM-1 (5.8 %, n=1), cpa1 (5.8 %, n=1) and cpa2 (5.8 %, n=1) were also detected. Conclusion. M13 PCR can be used in routine environmental surveillance for the detection of persistent antibiotic resistant clones in an ICU setting because of its reliability and simplicity. Further studies based on greater sample size, conducted at the multi-centre level, can give us a better understanding of the reliability of the molecular methods that can be used for the detection of persistent clones in the hospital setting.

Antimicrobial susceptibility testing (AST) is an important technique to find the susceptibility pattern of clinical isolates in order to administer the appropriate drug. One such technique is minimum inhibitory concentration (MIC), which not only identifies the right drug but also suggests the appropriate concentration necessary to neutralize the organisms in planktonic form. MIC can vary in case of adherent organisms since they form biofilms and activate survival mechanisms like quorum sensing. Here we have strategized a new method which used an inoculator plate, a resazurin dye, and a standard plate to identify minimum biofilm eradication concentration (MBEC) of adherent organisms.

Antimicrobial susceptibility testing (AST) is an important technique to find the susceptibility pattern of clinical isolates in order to administer the appropriate drug. One such technique is minimum inhibitory concentration (MIC), which not only identifies the right drug but also suggests the appropriate concentration necessary to neutralize the organisms in planktonic form. MIC can vary in case of adherent organisms since they form biofilms and activate survival mechanisms like quorum sensing. Here we have strategized a new method which used an inoculator plate, a resazurin dye, and a standard plate to identify minimum biofilm eradication concentration (MBEC) of adherent organisms.