1. Recombinant Viruses: Making Viruses Work For Us

2. Introduction
Viruses provide potent and constantly-evolving threats to human health, to animals and plants, they also offer the possibility of being used for human benefit through the construction and appropriate use of recombinant viruses.
These are viruses where the genome has been altered in a planned way by experimental manipulation.
A recombinant virus is a virus produced by recombining pieces of DNA using recombinant DNA technology

3. Uses Recombinant viruses have been invaluable in the study of:
Virus replication cycles, allowing us to study the effect on function of precisely targeted changes in the viral genome.
They are also powerful tools in the study of fundamental cell processes in the laboratory,
where they can be used to carry cDNA for specific host genes into cells in culture and hence to cause expression of the protein products.
Another use of recombinant viruses is as vaccines.
Finally, recombinant viruses have been of great interest as potential gene therapy agents.
The focus of intense research interest over the past 20 years

4. Generate a Recombinant Virus
First step to generate a recombinant virus is to clone its genome.
After genetic material has been isolated from virus particles, it can be manipulated in exactly the same way as any other RNA or DNA molecule.
DNA virus genomes may be cloned directly
While RNA virus genomes may be cloned as cDNA
These cloned molecules can then be modified by:
site specific alteration
or, portions may be removed and replaced with foreign DNA sequences
In this way, it is quite straightforward to generate modified forms of either an intact viral genome or, if the genome is large, a portion of it, cloned in a bacterial plasmid.

6. Generate a Recombinant Virus
What is far more difficult is to complete the process by using these cloned sequences to recreate infectious virus particles, a process often termed virus rescue.
This requires techniques specific to each type of virus and is not yet possible for all virus types.
At its simplest, all that is needed is to transfect the purified recombinant genome into susceptible cells.
However, many viruses also need some of their proteins to be supplied with the genome in order for any recombinant virus to be recovered.
Some of the double stranded RNA viruses remain refractory to all attempts at generating recombinant virus from cloned cDNA.

7. Generate a Recombinant Virus Vaccine
A number of genetically stable, safe live viral vaccines have been produced, e.g. measles and yellow fever.
Live vaccines have a number of advantages
but when attempting to expand the range of vaccines to include new targets it is often not possible to achieve a suitable live vaccine derivative of the pathogenic virus.
Recombinant viral vaccines offer a path to achieving the benefits of live vaccines for such difficult cases.
The principle is to embed one or more genes from the target pathogen into the genome of an established attenuated viral vaccine strain or other nonpathogenic viral genome
when the recombinant vaccine is administered, its genes encode proteins from the target pathogen and hence elicit protective immune responses against it.

9. Recombinant Viruses for Gene Therapy
Gene therapy is simply the introduction of DNA sequences into the cells of a patient with the aim of achieving a clinical benefit.
It was originally regarded as a way of restoring normal function in patients with specific inherited single gene defects, such as cystic fibrosis.
absence of a normal copy of the gene means that a specific protein function is missing, with severe physiological consequences.
By putting back a ‘good’ copy of the gene (normally as cDNA) into the patient’s cells, all these consequences should, in theory, be corrected.

10. Recombinant Viruses for Gene Therapy
The gene therapy concept has now grown to include a variety of possible applications, with the greatest number of clinical trials being in the field of cancer therapy.
When it comes to treating cancer by gene therapy, the goal is totally different from gene therapy for inherited conditions.
Rather than trying to restore normal function to the tumour cells, the objective is to kill them, or to cause them to undergo apoptosis – but to do so specifically, so that normal cells are not damaged.

11. Recombinant Viruses for Gene Therapy
DNA has to be carried across the membranes of cells and ultimately to reach the cell nucleus.
The cell membrane is a huge barrier to such a long, highly-charged molecule and, if it is endocytosed into a cell, it is then vulnerable to degradation in lysosomes.
By contrast, nucleic acid that is inside an infectious virus particle avoids these problems.
A recombinant virus (the vector) carrying a foreign gene (often referred to as a transgene) being able to penetrate the defenses of the cell before unloading its cargo.

12. Recombinant Viruses for Gene Therapy
What that cargo does to the cell subsequently is totally dependent on the gene(s) chosen; having carried the gene into the cell, the role of the virus is over.
This process of virus-mediated delivery of a gene into a cell is known as transduction.

13. Necessary or desirable features in a virus to be used as a gene therapy vector
The virus must be able to enter the desired human cell
It must be possible to generate recombinant forms of the virus that are deleted for one or more essential functions and so do not replicate in the patient.
Equally, it must be possible to grow these replication-defective recombinants in cell cultures in the laboratory.
In its deleted, replication-defective form, the virus must be safe to use in people.
In particular, it should not be able to recover replication-competence by recombining with other viruses that might be present in vivo.
The virus should deliver its genetic material to the nucleus in the form of DNA.

14. Necessary or desirable features in a virus to be used as a gene therapy vector
It should be possible to grow recombinant forms of the virus to high concentrations.
The replication-defective form of the virus should be able to accommodate a sufficient length of foreign DNA to be useful
If therapy is to be ‘permanent’, the transgene needs to persist in the cells indefinitely and to be inherited by daughter cells if the cell divides.
a viral vector DNA needs to achieve either integration into a chromosome of the cell
or else be capable of autonomous replication of its genome and partition during cell division.

15. Retroviral Vectors for Gene Therapy
Retrovirus vectors have all their normal coding sequences removed and replaced with a cDNA of interest.
The recombinant virus is then grown in a special cell line (packaging cells) which provides the viral proteins that are needed to form particles containing the recombinant genome
When used therapeutically,
The virus particles reverse transcribe their genome
and integrate the DNA copy, including the transgene, randomly into the host cell genome using enzymes included in the particle
But the absence of all viral genes from the particle means that no further events of the infectious cycle can occur.

17. Retroviral Vectors for Gene Therapy
Several diseases have been the target of clinical trials of retrovirus-mediated gene therapy, in particular two inherited immunodeficiencies:
adenosine deaminase (ADA) deficiency
And X-linked severe combined immunodeficiency disease (X-SCID).
Both conditions are characterized by a profound lack of T lymphocytes and hence a failure of both humoral and cell-mediated arms of the adaptive immune response
a buildup of dATP in all cells, which inhibits ribonucleotide reductase and prevents DNA synthesis, so cells are unable to divide. Since developing T cells and B cells are some of the most mitotically active cells, they are highly susceptible to this condition.

18. LASN: vector derived from Moloney murine leukemia virus

19. Adenovirus Vectors for Gene Therapy
Adenovirus vectors have been widely developed for gene therapy applications.
However, immune recognition of cells that have taken up the vector remains a problem.
Their most promising application so far is in cancer gene therapy where such responses are an advantage.

20. Replication of oncolytic adenovirus in tumor cells results in tumor cell killing (oncolysis) and release of tumor-associated antigens (TAAs) in the tumor milieu.
The released TAAs are then captured by antigen-presenting cells
This process, in turn, stimulates tumor-specific T cells and ultimately leads to generation of a strong antitumor immune response.

21. Oncolytic Viruses for Cancer Therapy
The virus should only be able to enter and infect tumour cells.
Identifying a gene within the virus genome whose products are:
essential for virus replication in normal cells
but which are unessential for growth in tumour cells.
A recombinant virus carrying loss-of-function mutations in that gene should replicate in and kill tumour cells specifically.

22. Oncolytic Viruses for Cancer Therapy
There has been considerable interest in making oncolytic adenoviruses tumour selective based upon the function of wild-type virus in inhibiting p53 activity.
Since many human tumours are p53-deficient it was argued that a mutant adenovirus that was unable to inactivate p53 should replicate selectively in p53-deficient tumours relative to the surrounding normal tissue.

23. Oncolytic Viruses for Cancer Therapy

24. Recombinant Viruses in the Laboratory
Modern cell biology research includes the testing of how function is altered when:
particular proteins are either added to the contents of cells (transient or stable overexpression)
or else removed from the cell (gene knock-out, or transient or stable RNA knockdown).
For stable over-expression, a favoured approach is to:
clone the relevant promoter cDNA into a retroviral vector,
transduce the selected cell line with the vector
and then to select for cells that have incorporated the retrovirus using a dominant selectable marker (such as puromycin resistance) which is also included in the vector.
It is a potent translational inhibitor in both prokaryotic and eukaryotic cells. Resistance to puromycin is conferred by the puromycin N-acetyl-transferase gene (pac) from Streptomyces.

26. Recombinant Viruses in the Laboratory
In the same way, stable knock-down at the RNA level can be achieved by transducing cells with a retroviral vector that encodes an appropriate shRNA construct to down-regulate the target gene via the miRNA pathway.
When only transient gene delivery is desired, adenovirus vectors have also been widely used.
A short hairpin RNA or small hairpin RNA (shRNA) that can be used to silence target gene expression via RNA interference (RNAi).

27. Schematic of RNA interference.
RNA interference can be initiated by introducing synthetic double-stranded siRNA or plasmid/viral vector encoding short hairpin (sh)RNA.
The shRNA is transcribed in the nucleus and exported to the cytoplasm where it is processed by Dicer into siRNA.
The siRNA associates with the multiprotein complex RISC and one strand is degraded
RISC: RNA-induced silencing complex