Recombineering-based Manufacturing of Engineered Viral Vectors for Research and Therapy

Summary

Recombineering, a recombination-mediated genetic engineering method, enables precise viral genome modifications without relying on enzymatic digestion. This approach is crucial for basic research, such as targeted deletions, and for developing engineered viruses used in oncolytic therapies, gene therapy, and genetic vaccines, expanding possibilities in both research and clinical applications.

Abstract

The increasing demand for engineered viral vectors in both basic and translational research has underscored the need for flexible, rapid, and scalable methods to generate recombinant DNA viruses. Strategies relying on restriction enzyme digestion and ligation are constrained by sequence-dependent limitations and time-consuming cloning steps. Here, we describe the recombination-mediated genetic engineering method (recombineering) that circumvents these limitations by enabling precise and seamless modifications of viral genomes in bacterial artificial chromosomes (BACs). This approach allows for efficient deletion, insertion, or substitution of coding or non-coding genetic elements, providing a powerful platform for high-throughput viral vector development. Recombineering is particularly valuable in basic research applications, such as the deletion or mutation of viral genes to investigate their function. More importantly, this methodology enables the generation of tens of recombinant viruses encoding distinct immunostimulatory or therapeutic payloads in parallel, making it exceptionally well-suited for the rapid preclinical evaluation of novel constructs. While this technology can be potentially implemented for any scientific purpose, this article focuses on the application of recombineering in two specific areas: the generation of oncolytic viruses based on herpes simplex virus, and the development of non-replicative adenoviral vectors for gene transfer. In conclusion, recombineering offers a versatile approach to viral genome engineering, significantly accelerating the pipeline from design to functional testing. Its relevance spans from fundamental virology to translational medicine to meet evolving research and clinical needs.

Introduction

Viral vectors have emerged as potent tools in gene therapy, oncolytic virotherapy, and genetic vaccine development due to their natural ability to deliver genetic material into host cells. They leverage viruses' natural tropism and transduction mechanisms, facilitating both transient and long-term gene expression. They are able to survive in the extracellular environment, to attach to a specific cellular receptor (that defines the viral tropism) and promote their internalization, to hijack the host cell gene expression machinery and drive the expression of their own genes, and to elude membrane-bound and intracellular sensors of the innate immune arm

Protocol

As a prerequisite to recombineering, a proficient bacterial strain (e.g., SW102) with a BAC-Genome of Interest must be available. Although recombineering is a highly efficient method to enrich colonies that have undergone the desired modification, here, we describe a two-step recombineering strategy, referred to as the first and second steps (Figure 1). In the first step, an expression cassette containing both positive and negative selection markers is inserted into the locus of interest. In the second step, this cassette is replaced with the actual genetic modification intended for the final genome. The selection markers used in this stu....

Results

Generation of an IL12-coding oncolytic Herpes virus
As outlined in the introductory section of this work, one potential application of recombineering is the insertion of immunostimulatory transgenes into the genome of oncolytic viruses, with the aim of enhancing their immunotherapeutic potential. Below, we present an example of such an application using an oncolytic vector based on HSV-1. Specifically, this study investigates the impact of a transgene insertion into an intergenic locus within the Us1.......

Discussion

This "RECOMBINEERING" (homologous RECOMBInation-mediated genetic engiNEERING) protocol uses a two-step strategy: first, integration of a selection cassette into the genomic locus of interest, and second, replacement of this cassette with the desired modification (Figure 1).

The selection markers used include ampicillin resistance (AmpR), beta-galactosidase (β-gal), and sacB. AmpR enables antibiotic resistance-based selection, β-gal allows blue/white scre.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ampicillin	any	any	molecular grade
For oligo to amplify Il-12: CTGGCTAGCGTTTAA ACGGGCCCTCTAGACTCG AGCGGCCGCACGCCACCA TGTGTCCTCAGAAGCTAACC	any	any	HPLC purified
For oligo to amplofy AmpR/LacZ/SacB  cassette: CTGGCTAGCGTTTAAA CGGGCCCTCTAGACTCGAG CGGCCGCACGCCACCACC CCTATTTGTTTATTTTTC	any	any	HPLC purified
IPTG	any	any	molecular grade
LB agar
LB medium
Phusion Hot Start II High-Fidelity DNA Polymerase	Thermo Scientific	F549S
Rev oligo to amplify Il12: GGCAACTAGAAGGCA CAGTCGAGGCTGATCAGC GGTTTAAACTTAAGCTTTC AGGCGGAGCTCAGATAG	any	any	HPLC purified
Rev oligo to amplofy AmpR/LacZ/SacB  cassette: GGCAACTAGAAGGCAC AGTCGAGGCTGATCAGC GGTTTAAACTTAAGCTTT TATTTGTTAACTGTTAATTG	any	any	HPLC purified
SacB/AmpR/LacZ selection cassette	this manuscript	-	-
Sucrose	any	any	molecular grade
SW102 with BAC-HSV-1 or BAC-Adv	this manuscript	-	-
Tryptone	any	any	molecular grade
Wizard SV Gel	Promega	A9281
X-gal	any	any	molecular grade
Yeast extract	any	any	molecular grade