A Novel Gene Editing Strategy: Circular ssDNA Significantly Enhances Efficiency in Hematopoietic Stem Cell Gene Therapy

Introduction: The “Template Battle” in Gene Therapy

Gene therapy, particularly strategies based on hematopoietic stem and progenitor cells (HSPCs), is hailed as a beacon of hope for curing inherited blood disorders. However, the success of gene editing has long depended heavily on the delivery of “gene templates.”

The mainstream template carrier to date has been adeno-associated virus (AAV), especially AAV6. While efficient, it comes with a host of issues: genotoxicity, impaired engraftment, loss of editing events, high production costs, and limited cargo capacity.

In recent years, non-viral templates have stepped into the spotlight. From linear double-stranded DNA (LdsDNA) to linear single-stranded DNA (LssDNA), scientists have continuously experimented, yet struggled to overcome the bottlenecks of “low efficiency and high toxicity.” It wasn’t until the emergence of circular single-stranded DNA (CssDNA) that a true breakthrough became visible.

Why is CssDNA So Special?

CssDNA, as the name suggests, is a single-stranded DNA molecule with a circular structure. Compared to linear DNA, it boasts three natural advantages:

1. High Nuclease Resistance: Less susceptible to degradation by cytoplasmic exonucleases;

2. Low Immunogenicity: Minimizes activation of intracellular DNA-sensing pathways (e.g., cGAS-STING);

3. Structural Stability: The circular form facilitates nuclear entry post-electroporation.

These properties make CssDNA a theoretically ideal non-viral template for gene editing. This study is the first to systematically validate its performance in HSPCs.

Study Design: CssDNA vs. LssDNA vs. AAV6

The research team designed a comprehensive gene insertion workflow using TALEN technology to cleave target gene loci (e.g., B2M) and compared CssDNA, LssDNA, and AAV6 as donor templates.

Key experiments included:

• Multi-length Template Testing: From 0.6 kb to 2.2 kb, verifying CssDNA’s suitability for long-fragment gene insertion;

• Multi-locus Editing: Testing at B2M, AAVS1, CD11b, S100A9, and other loci;

• Functional Validation: Assessing cell viability, differentiation, and long-term engraftment via in vitro colony-forming unit (CFU) assays and in vivo transplantation in NCG mouse models;

• Single-cell CITE-seq Analysis: Deep molecular phenotyping of edited cells.

Key Findings: CssDNA Outperforms Across the Board

1. Dramatically Enhanced Editing Efficiency

• CssDNA achieved knock-in (KI) efficiencies of 40%–49%, 3- to 5-fold higher than LssDNA;

• Stable performance across multiple gene loci demonstrates broad applicability;

• Higher KI/KO ratios indicate a preference for precise integration over error-prone repair.

2. Preserved Cell Viability and Differentiation

• Although electroporation itself caused some damage, CssDNA did not exacerbate toxicity;

• Edited HSPCs retained normal differentiation capacity into erythroid, myeloid, and other blood lineages.

3. Superior In Vivo Engraftment

• In NCG mice, CssDNA-edited HSPCs showed higher engraftment levels and more stable maintenance of gene edits;

• Compared to the AAV6 group, the CssDNA group had 5 times more KI(+) cells, with edits remaining stable long-term.

Mechanism Unveiled: Why is CssDNA Better?

Single-cell transcriptomics revealed the biological mechanisms behind CssDNA’s advantages:

• More Primitive Stem Cells Edited: The CssDNA group had 5 times more long-term hematopoietic stem cells (LT-HSCs) than the AAV group, with 10 times more KI(+) LT-HSCs;

• More Quiescent Metabolic State: CssDNA-edited LT-HSCs were predominantly in G0/G1 phase, with lower metabolic activity, favoring long-term engraftment;

• Enhanced Bone Marrow Homing: Upregulation of niche adhesion markers like CXCR4, CD44, and FIIR;

• Controlled Inflammatory Response: Although CssDNA triggered interferon pathways, this response was transient and reversible, not compromising cell function.

Broader Applications: CssDNA in T Cell Engineering

The team also applied the CssDNA/TALEN system to primary T cells, showing:

• KI efficiencies up to ~40%, far exceeding LssDNA;

• Cell viability maintained above 90%, with preserved expansion capacity.

This opens new avenues for immunotherapies like CAR-T.

Future Outlook: CssDNA Leading the Next Generation of Gene Therapies

This study not only confirms the high efficiency and safety of CssDNA in HSPC editing but also highlights its potential as a universal, scalable, non-viral gene editing template.

Looking ahead, we can anticipate:

• Integration of CssDNA with various gene editors (e.g., CRISPR-Cas9, TALEN, Cas12a);

• Further reduction of immunogenicity via chemical modifications;

• Optimization of scalable production processes for GMP-compliant clinical use.

Conclusion

From AAV to LssDNA, and now to CssDNA, a “template revolution” in gene therapy is quietly underway. This research not only provides a superior gene insertion strategy but also opens new doors for treating genetic diseases, cancers, and immune disorders.

CssDNA may well be the key we’ve been searching for.