$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
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 arm1,2. In this way, after a productive viral infection, viruses transform cells into viral progeny factories that spread new viruses into the organism, eventually causing a disease.
Wild-type viruses can be transformed into a biotechnological tool by genetic engineering: their genome can be easily manipulated to retain the transduction capability and obtain a clinical outcome. Based on their clinical application, viral vectors can be engineered to retain (at least in part) or be ablated in their replication competence to generate either replication-competent or replication-incompetent vectors3,4.
Replication-competent viruses are mainly intended for oncolytic virus purposes. Cancer cells, often deficient in antiviral defenses1, allow replication of attenuated recombinant viruses engineered through deletion of virulence genes. These viruses selectively replicate in tumors while sparing normal tissue. As understanding of virology has advanced, attenuated viruses have been superseded by more precise genome modifications designed to enhance both safety and therapeutic efficacy.
Among the fields of interest requiring surgical molecular engineering of viral genomes are: i) Replication-conditional oncolytic viruses, where viral promoters are replaced with tumor-associated promoters (cellular promoters over-transcribed in tumor cells), thereby restricting viral gene expression to diseased cells5. ii) Viruses with altered tropism6,7, where envelope glycoproteins that determine infection can be modified or replaced with antibody fragments or ligands that target proteins overexpressed on tumor cells8,9. iii) Armed oncolytic viruses. Tumor cell death induced by oncolytic viruses triggers immunogenic cell death (ICD), characterized by the release of inflammatory mediators1,10 and tumor antigens11. These signals promote efficient recognition of tumor antigens by the immune system, restoring surveillance12and enabling combination with immune checkpoint inhibitors13,14,15 and other immune therapeutics such as RNA-based therapies and vaccines16.
In support of this immunotherapeutic effect, transgenes of interest can be encoded within the viral genome, thereby exploiting the virus not only for its oncolytic activity but also to enhance its immunotherapeutic potential through the in-situ production of relevant payloads3,7,17,18,19. These payloads may comprise cytokines, chemokines, antibodies, suicide genes, and antigens3,20. Insertion requires precision, with intergenic regions of the viral genome preferred to minimize disruption of viral genes and preserve replication and potency7. Therefore, techniques that enable the screening of both optimal insertion sites and transgenes of interest are essential.
On the other hand, in the context of non-replicative viruses, used for gene therapy or as vehicles for genetic vaccines, it is important that the viral vector delivers the transgene (either transiently or long-lasting) without replicating into the host cell to avoid toxicity21. Adenoviral vectors are characterized by high transduction efficiency, robust expression of transgenes, and the capacity to infect both dividing and non-dividing cells3. These properties have made them first-in-class for the development of genetic vaccines, including those for emerging infectious diseases, such as COVID-19, as well as gene replacement therapies22. The most famous example of an adenoviral vector that has reached the market is the Oxford-AstraZeneca COVID-19 vaccine, known by the commercial name of Covishield or Vaxzevria, based on a modified chimpanzee adenovirus, ChAdOx1, encoding for the Spike protein of SARS-CoV-2. When injected by the intramuscular route, Covishield is able to penetrate inside the cells and induce the expression of the Spike protein, priming the immune system to mount an immune response against it3.
There is a clear need to modify viral genomes without being constrained by restriction enzyme recognition sites. Among oncolytic viruses, Herpes Simplex Virus-1 (HSV-1)-based vectors offer unique advantages, including lytic ability, safety, and large genome size, allowing insertion of sizeable transgenes or multiple genetic elements23,24. Beyond HSV, many other viruses show therapeutic promise, as recently reviewed in Sasso et al.3. Rather than a universal "best-in-class" vector, different viral platforms are suited to distinct therapeutic needs3. Currently, homologous recombination systems in bacteria or hybrid yeast-bacteria cloning remain the most common alternatives to restriction enzymes, but these are laborious, multi-step, and inefficient25.
An emerging alternative is the use of CRISPR/Cas9, recently applied to engineer genomes of viruses, including vaccinia virus (VV), herpes simplex virus, and adenoviral vectors. Cas9 is appealing for gene inactivation via indel mutations but becomes less practical when multiple cuts are required to delete genome segments or insert a transgene via donor DNA. Moreover, editing is limited by gRNA design, off-target effects in both viral and host genomes, and dependence on PAM sequences in regions of interest25.
With this in mind, and through two examples based on HSV and adenovirus, this article illustrates the recombineering method, which can be applied broadly-from viruses to bacteriophages, complex plasmids, and diverse experimental systems. Recombineering (homologous RECOMBInation-mediated genetic engiNEERING) is an in vivo genetic engineering technique, used primarily in Escherichia coli, that exploits temperature-dependent bacteriophage recombination proteins to catalyze homologous recombination between DNA elements with as few as 30 bp of homology26. This approach allows precise insertion, deletion, or modification of sequences without dependence on restriction sites or DNA size. In this manuscript, we describe a version based on the heat-inducible E. coli strain SW102, though alternative strains with specific inducible systems are also available27.
Recombineering begins with preparing electrocompetent bacterial cells through a controlled temperature shock to activate phage recombination machinery. DNA molecules carrying the appropriate homology arms are then electroporated. These may be double-stranded DNAs (dsDNA) generated by restriction digestion or PCR or single-stranded DNAs (ssDNAs) such as oligonucleotides. The presence of homologous sequences together with active recombination proteins enables the creation of new DNA molecules with the desired modification.
Recombineering molecular cloning technology enables precise genetic engineering and is applied in diverse contexts, including insertion of selectable or non-selectable markers in plasmids, bacterial chromosomal DNA, and bacterial artificial chromosomes (BACs). BACs are particularly valuable for cloning large genomic fragments, including adenoviral and HSV DNA, enabling the rapid production of viral vectors. Beyond translational applications, recombineering also supports basic research by permitting targeted deletion, modification, or replacement of viral genes or protein domains. Recombineering has some limitations: short homology arms hinder recombination in repetitive regions; PCR-amplified substrates may occasionally introduce mutations; and the sequence of the target region must be known.