Abstract
Most systemic viral gene therapies have been limited by sequestration and degradation of virions, innate and adaptive immunity, and silencing of therapeutic genes within the target cells. Here we engineer a high-affinity protein coat, shielding the most commonly used vector in clinical gene therapy, human adenovirus type 5. Using electron microscopy and crystallography we demonstrate a massive coverage of the virion surface through the hexon-shielding scFv fragment, trimerized to exploit the hexon symmetry and gain avidity. The shield reduces virion clearance in the liver. When the shielded particles are equipped with adaptor proteins, the virions deliver their payload genes into human cancer cells expressing HER2 or EGFR. The combination of shield and adapter also increases viral gene delivery to xenografted tumors in vivo, reduces liver off-targeting and immune neutralization. Our study highlights the power of protein engineering for viral vectors overcoming the challenges of local and systemic viral gene therapies.
Highlights
Most systemic viral gene therapies have been limited by sequestration and degradation of virions, innate and adaptive immunity, and silencing of therapeutic genes within the target cells
The development of ‘gutless’ AdVs, which are devoid of viral genes and extend safety margins even further, has enabled the utilization of up to 35 kilobase pairs for transgene expression, which exceeds the capacity of associated virus (AAV) vectors by one order of magnitude[9]
If factor X (FX)-shielded virions enter the cytosol, FX can act as a pathogen-associated molecular pattern (PAMP) and trigger innate immunity against the infected cells, which in turn may reduce the lifetime of the transduced cells[22]
Summary
Most systemic viral gene therapies have been limited by sequestration and degradation of virions, innate and adaptive immunity, and silencing of therapeutic genes within the target cells. The development of ‘gutless’ AdVs, which are devoid of viral genes and extend safety margins even further, has enabled the utilization of up to 35 kilobase pairs (kb) for transgene expression, which exceeds the capacity of AAV vectors by one order of magnitude[9] This will allow the delivery of multiple payload genes at once, and potentially the secretion of a cocktail of therapeutic proteins upon delivery of the non-oncolytic vector to a tumor[10], without vector replication and with enhanced safety. Likewise, shielding attempts with engineered protein coats showed promising results in vitro, but were ineffective in vivo[25] Besides overcoming detargeting, another challenge for viral tumor gene therapy is the targeting of the vector to the cancer cells. Virion binding to CAR and integrin receptors triggers endocytosis and escape to the cytosol from non-acidic early endosomes[32]
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