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Near-Perfect Precise Genome Editing in Primary Human Hematopoietic Stem and Progenitor Cells

Core Concepts
Optimized protocols combining guide RNA delivery, donor design, and small molecule additives can achieve near-perfect (>90%) precise editing efficiencies in primary human hematopoietic stem and progenitor cells with minimal toxicity and no observed off-target effects.
The authors developed an optimized protocol for precise genome editing in primary human hematopoietic stem and progenitor cells (HSPCs) that can achieve near-perfect (>90%) editing efficiencies. Key elements of the protocol include: 48-hour pre-stimulation of HSPCs in growth factors and UM171 to induce cell cycling Use of the DNA-PK inhibitor AZD7648 to enhance homology-directed repair (HDR) efficiency Inclusion of silent mutations in the donor DNA to disrupt the spacer sequence in addition to the PAM sequence Optimization of ribonucleoprotein (RNP) concentration and delivery Addition of p53 siRNA to improve cell survival The authors tested both adeno-associated virus (AAV) and short single-stranded oligodeoxynucleotide (ssODN) donors, and found that the ssODN donors could achieve similar near-perfect editing efficiencies when designed with the spacer-disrupting silent mutations. Importantly, the editing was found to be even across the hematopoietic hierarchy, with no significant effects on progenitor phenotypes, lineage outputs, or the frequency of high self-renewal potential long-term culture-initiating cells. The authors also demonstrated the ability to tune the zygosity of edited cells by providing a mixture of mutant and wild-type donor DNA. This enables the protocol to be useful for both therapeutic editing strategies and disease modeling applications. Overall, this optimized protocol represents a significant advance in the field of precise genome editing in primary human HSPCs, opening new avenues for both curative treatments and accurate disease modeling.
The mean precise editing efficiencies achieved were >90% for both AAV and short ssODN donors. The addition of 0.5 μM AZD7648 increased HDR efficiency up to 80-90% in primary human HSPCs. The combination of AZD7648, p53 siRNA, and optimized RNP and donor conditions achieved mean editing efficiencies of 97% with minimal toxicity.
"Combining AZD7648 with optimal pre-stimulation, RNP, p53 siRNA and AAV donor concentrations yielded mean efficiencies of 97% editing with minimal toxicities." "Surprisingly, these conditions worked at equivalent efficiencies for short single-stranded oligodeoxynucleotide donors (ssODNs) giving mean efficiencies of 94% when the ssODNs were modified to mutate not just the PAM sequence but also multiple positions in the spacer (silent mutations)."

Deeper Inquiries

How might the optimized editing protocol be leveraged to develop curative gene therapies for monogenic blood disorders?

The optimized editing protocol outlined in the study can be instrumental in developing curative gene therapies for monogenic blood disorders by significantly enhancing the efficiency of precise genome editing in human hematopoietic stem and progenitor cells (HSPCs). With mean precise editing efficiencies exceeding 90%, the protocol allows for the correction of disease-causing mutations with near-perfect accuracy. This high level of efficiency is crucial for ensuring that the desired genetic modifications are successfully integrated into the target cells, leading to the potential for curative treatments. By utilizing this protocol, researchers and clinicians can target specific genetic mutations responsible for monogenic blood disorders, such as sickle cell anemia or thalassemia, and replace them with the correct sequences. This precise editing capability opens up new avenues for developing personalized gene therapies that address the root cause of these disorders at the genetic level, offering the possibility of long-term and potentially permanent cures for affected individuals.

What are the potential limitations or safety concerns associated with the use of DNA-PK inhibitors like AZD7648 for enhancing genome editing in HSPCs?

While DNA-PK inhibitors like AZD7648 have shown promise in enhancing genome editing efficiency in HSPCs, there are potential limitations and safety concerns that need to be considered. One primary concern is the off-target effects of DNA-PK inhibitors, as these molecules can impact the DNA repair pathways in cells, potentially leading to unintended genetic alterations at sites other than the target locus. It is essential to thoroughly evaluate the specificity of DNA-PK inhibitors to minimize off-target effects and ensure that the editing process remains precise and controlled. Additionally, the long-term effects of DNA-PK inhibition on the overall genomic stability and functionality of edited HSPCs need to be carefully monitored. There may be risks associated with perturbing the DNA repair mechanisms in these stem cells, which could impact their regenerative capacity and long-term engraftment potential. Furthermore, the optimal dosage and duration of DNA-PK inhibitor treatment must be determined to balance the desired editing efficiency with potential cytotoxicity or adverse effects on cell viability. Close attention to these safety considerations is essential to ensure the successful and safe application of DNA-PK inhibitors in genome editing approaches for HSPCs.

Could the principles of donor design and small molecule optimization demonstrated here be applied to improve precise genome editing in other primary cell types beyond HSPCs?

Yes, the principles of donor design and small molecule optimization demonstrated in the study can be applied to improve precise genome editing in other primary cell types beyond HSPCs. The key factors identified, such as the incorporation of silent mutations in the donor design, the use of specific small molecule inhibitors like DNA-PK inhibitors, and the optimization of delivery methods, are fundamental aspects of enhancing genome editing efficiency in various cell types. By tailoring the donor design to include spacer-breaking silent mutations and optimizing the use of small molecule additives, researchers can potentially improve the editing efficiency in different primary cell populations. These principles can be adapted and applied to other cell types, such as induced pluripotent stem cells (iPSCs), neural stem cells, or mesenchymal stem cells, to enhance the precision and efficacy of genome editing for diverse therapeutic and research applications. The knowledge gained from this study can serve as a foundation for developing optimized editing protocols in a wide range of primary cell types, paving the way for more effective and targeted genome editing strategies in various biological contexts.