crisprcas9-gene-editing-hematopoietic-stem-progenitor-cells-for-gene

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene ...

Christian Medical College Campus

Intracellular delivery of nucleic acids is one of the critical steps in the transfections. Prior findings demonstrated various strategies including membrane fusion, endosomal escape for the efficient cytoplasmic delivery. In our continuing efforts to improve the nucleic acids transfections, we harnessed cell permeable properties of Tomatidine (T), a steroidal alkaloid abundantly found in green tomatoes for maximizing intracellular delivery of lipoplexes. We doped Tomatidine into liposomes of cationic lipid with amide linker (A) from our lipid library. Six liposomal formulations (AT) of Lipid A (1 mM) with varying concentrations of Tomatidine (0-1 mM) were prepared and evaluated for their transfection efficacies. Owing to its signature characteristic of cell membrane permeability, Tomatidine modulated endocytosis process, enhanced the intracellular delivery of the lipoplexes, and in turn increased the transfection efficacy of cationic liposomes. Our findings provide 'proof of concept' for enhancing transfections in gene delivery applications with Tomatidine in cationic liposomal formulations. These findings can be further applied in lipid mediated gene therapy and drug delivery applications.

Exploring membrane permeability of Tomatidine to enhance lipid mediated nucleic acid transfections.

Cationic lipid-guided nucleic acid delivery holds great promise in gene therapy and genome-editing applications for treating genetic diseases. However, the major challenge lies in achieving therapeutically relevant efficiencies. Prior findings, including our own, demonstrated that asymmetry in the hydrophobic core of cationic lipids imparted superior transfection efficiencies. To this end, we have developed a lipid nanocarrier system with an asymmetric hydrophobic core () derived from a mixture of fatty acids of food-grade palmstearin and compared its efficiency with symmetric palmitic acid-based nanocarrier system ().  exhibited superior transfection efficiencies with both plasmid DNA (pDNA) and mRNA in multiple cultured cells than the control . More importantly,  exhibited 2-fold superior transfections with linear nucleic acid, green fluorescent protein (GFP) mRNA in hematopoietic cells, when compared with the commercial control lipofectamine RNAiMAX.  was also found to be effective in delivering genome-editing tools (CRISPR/Cas9, sgRNA encoded pDNA with a reporter GFP construct) than  in HEK-293 cells. In the present study, we report that cationic liposomes derivatized from natural food-grade fat palmstearin with a natural hydrophobic core asymmetry are efficient in delivering both linear and circular nucleic acids. In particular,  is efficient in delivering mRNA to hematopoietic cells. These findings can be further exploited in the genome-editing approach for treating β-globinopathies.

Green Transfection: Cationic Lipid Nanocarrier System Derivatized from Vegetable Fat, Palmstearin Enhances Nucleic Acid Transfections.

The ability to precisely change the deoxyribonucleic acid (DNA) bases at specific sites offers tremendous advantages in the field of molecular biology and medical biotechnology. Identification of Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR), revelation of its role in prokaryotic adaptive immunity and subsequent conversion into genome and epigenome engineering system are the landmark research progresses of the decade. The possibilities of deciphering the molecular mechanisms of the disease, identifying the disease targets, generating the disease models, validating the drug targets, developing resistance to the infection and correcting the genotype have brought off much enthusiasm in the field of infectious diseases and genetic disorders. This review focuses on CRISPR/Cas9's impact in the field of infection and genetic disorders.

CRISPR-Cas9 Probing of Infectious Diseases and Genetic Disorders.

β-Hemoglobinopathies are the most common monogenic disorders, and a century of research has provided us with a better understanding of the attributes of these diseases. Allogenic stem cell transplantation was the only potentially curative option available for these diseases until the discovery of gene therapy. The findings on the protective nature of fetal hemoglobin in sickle cell disease (SCD) and thalassemia patients carrying hereditary persistence of fetal hemoglobin (HPFH) mutations has given us the best evidence that the cure for β-hemoglobinopathies remains hidden in the hemoglobin locus. The detailed understanding of the developmental gene regulation of gamma-globin (γ-globin) and the emergence of gene manipulation strategies offer us the opportunity for developing a γ-globin gene-modified autologous stem cell transplantation therapy. In this review, we summarize different therapeutic strategies that reactivate fetal hemoglobin for the gene therapy of β-hemoglobinopathies.

Manipulation of Developmental Gamma-Globin Gene Expression: an Approach for Healing Hemoglobinopathies.

HIV infection continues to be a serious health issue with an alarming global spread, owing to the fact that attempts at developing an effective vaccine or a permanent cure remains futile. So far, the only available treatment for the clinical management of HIV is the combined Anti-Retroviral Therapy (cART), but the long-term cART is associated with metabolic changes, organ damages, and development and transmission of drug resistant HIV strains. Thus, there is a need for the development of one-time curative treatment for HIV infection. The allogeneic transplantation with the Hematopoietic Stem and Progenitor cells (HSPCs) having 32 bp deletion in Chemokine receptor 5 gene (CCR5 Δ32) demonstrated successful HIV remission in the Berlin and London patients, and highlighted that transplantation of CCR5 null HSPCs is a promising approach for a long- term HIV remission. The advent of gene editing technologies offers a new choice of generating ex vivo CCR5 ablated allogeneic or autologous HSPCs for stem cell transplantation into HIV patients. Many groups are attempting CCR5 disruption in HSPCs using various gene-editing strategies. At least two such studies, involving CCR5 gene editing in HSPCs have entered the clinical trials. This review aims to outline the strategies taken for CCR5 gene editing and discuss the challenges associated with the development of CCR5 manipulated HSPCs for the gene therapy of HIV infection.

The Strategies and Challenges of CCR5 Gene Editing in Hematopoietic Stem and Progenitor Cells for the Treatment of HIV.

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.

Preferential Expansion of Human CD34CD133CD90 Hematopoietic Stem Cells Enhances Gene-Modified Cell Frequency for Gene Therapy.

The advent of next-generation genome engineering tools like CRISPR-Cas9 has transformed the field of gene therapy, rendering targeted treatment for several incurable diseases. Hematopoietic stem and progenitor cells (HSPCs) continue to be the ideal target cells for gene manipulation due to their long-term repopulation potential. Among the gene manipulation strategies such as lentiviral gene augmentation, non-homologous end joining (NHEJ)-mediated gene editing, base editing and prime editing, only the homology-directed repair (HDR)-mediated gene editing provides the option of inserting a large transgene under its endogenous promoter or any desired locus. In addition, HDR-mediated gene editing can be applied for the gene knock-out, correction of point mutations and introduction of beneficial mutations. HSPC gene therapy studies involving lentiviral vectors and NHEJ-based gene-editing studies have exhibited substantial clinical progress. However, studies involving HDR-mediated HSPC gene editing have not yet progressed to the clinical testing. This suggests the existence of unique challenges in exploiting HDR pathway for HSPC gene therapy. Our review summarizes the mechanism, recent progresses, challenges, and the scope of HDR-based gene editing for the HSPC gene therapy.

Homology-directed gene-editing approaches for hematopoietic stem and progenitor cell gene therapy.

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.

Saravanabhavan Thangavel

Centre for Stem Cell Research (CSCR),
A unit of InStem Bengaluru,
Centre for Stem Cell Research (CSCR), A unit of InStem Bengaluru

Christian Medical College Campus

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

Saravanabhavan Thangavel

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Christian Medical College Campus

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

Centre for Stem Cell Research (CSCR),
A unit of InStem Bengaluru,
Centre for Stem Cell Research (CSCR), A unit of InStem Bengaluru