Researchers from San Raffaele Hospital have developed innovative approaches to significantly enhance the potency of lentiviral vector (LV) gene therapy targeting liver cells, potentially transforming treatment options for genetic liver disorders including hemophilia.
The study, published in Nature Communications, demonstrates multiple strategies to boost the efficiency of gene delivery to hepatocytes both before and after vector administration, achieving remarkable increases in therapeutic protein production.
Enhancing Gene Therapy Potency: Before and After Approaches
The research team, led by Canepari and colleagues, explored two distinct approaches to improve lentiviral vector performance: enhancing transduction efficiency at the time of administration (a priori) and expanding genetically modified hepatocytes after treatment (a posteriori).
"We successfully enhanced the potency of in vivo LV-mediated hepatocyte gene transfer, thus paving the way to expanding its in vivo applicability and easing its clinical translation," the researchers stated in their paper.
For the a posteriori approach, the team exploited a selective advantage strategy using lentiviral vectors carrying an anti-Cypor shRNA alongside therapeutic transgenes encoding clotting factors. When mice were subsequently treated with acetaminophen, the genetically modified hepatocytes were protected from toxicity, allowing them to expand while unmodified cells were damaged.
This selection process resulted in up to 50-fold increases in circulating factor IX (FIX) levels in mice receiving low initial vector doses. Importantly, the selection could be halted and resumed on demand, offering potential control over therapeutic protein levels.
Three Key Transduction Enhancers Identified
For the a priori approach, the researchers identified three minimally invasive interventions that significantly improved hepatocyte transduction:
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Proteasome inhibition: A single dose of Bortezomib (Velcade) administered one hour before LV injection resulted in 2-3 fold higher transgene output for FIX and 10-30 fold higher output for factor VIII (FVIII).
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Type-I interferon signaling blockade: Pre-treatment with an anti-IFNAR1 antibody increased FVIII output by 20-40 fold in hemophilia A mice.
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Fasting regimen: A 24-hour fast before vector administration enhanced transgene output approximately 3-fold, likely through modulation of the low-density lipoprotein receptor (LDLR) pathway.
When these approaches were combined, the researchers achieved up to 13-fold higher transgene output compared to standard LV administration. Most notably, combining fasting and Bortezomib with phagocytosis-shielded LVs resulted in a remarkable 40-fold increase in FIX levels.
Potential for Clinical Translation
The researchers emphasized that all three transduction enhancement strategies are potentially clinically applicable:
"All the proposed strategies to enhance LV transduction a priori are transient (fasting) or single-dose and potentially clinically applicable," they noted, pointing out that Bortezomib is already approved for multiple myeloma, anti-IFNAR1 antibody (Anifrolumab/Saphnelo) is used for lupus, and short-term fasting is feasible in humans.
The study demonstrated that these approaches could achieve therapeutic levels of clotting factors in hemophilia models while using lower vector doses, potentially reducing manufacturing costs and dose-dependent toxicities.
Mechanistic Insights
The research provided several mechanistic insights into how these interventions enhance gene transfer:
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Bortezomib appears to work through both direct effects on hepatocytes and indirect effects by reducing transduction of liver immune cells, making more vector available for hepatocytes.
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Surprisingly, the absence or reduction of the low-density lipoprotein receptor (LDLR) improved rather than impaired LV transduction, suggesting that fasting enhances transduction partly through LDLR downregulation.
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The combination of transduction enhancers with phagocytosis-shielded LVs showed synergistic effects, indicating complementary mechanisms of action.
Safety Considerations
The researchers addressed potential safety concerns, particularly for the a posteriori selection approach, which involves clonal expansion of transduced hepatocytes. Integration site analysis showed no enrichment of vector integrations near cancer-associated genes in mice undergoing selection.
However, one mouse developed a hepatocellular adenoma during the selection procedure, highlighting potential risks of chronic hepatotoxic regimens. The researchers suggested that the a priori enhancement strategies might be preferable for many applications, as they avoid prolonged liver damage.
Future Directions
This work opens several avenues for future research and clinical development:
"Our work highlights the translational potential of a minimally invasive, cost-effective combination of treatments, which could be employed together with LV to enhance the potency of in vivo gene therapy to hepatocytes, enabling to tackle new and more challenging diseases requiring a high percentage of hepatocyte transduction," the authors concluded.
The ability to achieve higher transgene expression with lower vector doses could expand the range of treatable conditions and reduce manufacturing challenges that have limited the clinical application of lentiviral vectors for in vivo gene therapy.
These findings represent a significant step forward in making lentiviral vector-based gene therapy more effective and accessible for patients with genetic liver disorders.
