Rapid method for construction of recombinant HSV gene transfer vectors

Area of Science:

• Virology
• Molecular Biology
• Gene Therapy

Background:

• Herpes simplex virus type 1 (HSV-1) is a neurotrophic virus that establishes lifelong latency in neurons.
• HSV-1 possesses a large genome encoding numerous viral functions, making it a potential candidate for gene therapy vectors.
• Efficient methods for genetic manipulation of HSV-1 are crucial for developing effective gene therapy applications.

Purpose of the Study:

• To develop and validate a two-step method for insertion/deletion mutagenesis of HSV-1 genes.
• To enable the efficient insertion of foreign transgenes into specific locations within the HSV-1 genome.
• To create versatile HSV-1-based vectors for gene therapy applications.

Main Methods:

• A two-step mutagenesis strategy was employed involving reporter gene insertion and subsequent transgene substitution.
• The first step involved inserting a lacZ reporter gene cassette flanked by PacI sites into target HSV-1 genes.
• The second step utilized PacI digestion and homologous recombination to replace the lacZ gene with foreign cDNAs, generating recombinant viruses.

Main Results:

• The method achieved high frequencies of recombinant virus formation (80-100% for potential recombinants).
• Transgene insertion efficiency ranged from 19-65% among the identified recombinants.
• The procedure allowed for sequential deletion and insertion of multiple genes, demonstrated by creating a vector with two expression cassettes.

Conclusions:

• A robust and efficient two-step method for HSV-1 genome engineering was established.
• This technique facilitates the creation of HSV-1 vectors with precisely inserted transgenes for gene therapy.
• The developed method supports sequential genetic modifications, enabling the construction of complex, multi-gene modified viral vectors.