Viral vectors; our friends, not foes!

The word ‘virus’ recalls unpleasant thoughts of maladies like small pox, rabies, AIDS, Ebola, flu (no need to say, nowadays COVID-19) that killed millions of people in the history of mankind. Not all viruses are villains that cause deadly diseases, some even help us to cure illness (yes, seriously!). By utilizing cutting edge molecular biology techniques, we can modify even those deadly viruses to harmless tools for specific research and medical applications. Such modified viruses (here onwards will write as viral vectors) are apt ‘cargo-vans‘ to deliver foreign genes into host cells. Recombinant virus technology has unique features, establishing it as one of the most efficient techniques to address several scientific questions. It may become ´the therapeutic tool´ for fixing genetic disorders in future clinics.

Before going farther into the viral vectors, let’s talk about the less conveyed ´good side´ of viruses. Do you know a good portion of human genome consists of viral genomes? A type of viruses, especially retroviruses (for e.g. HIV, that causes AIDS belongs to the same family) while infecting the host cells, integrate their DNA into the host cell´s DNA and may remain silent for long time. When appropriate condition arises (like cases of immune suppression in host) viral DNA wakes up from the dormant stage and start forming infectious viral particles which can spread the infection further. These DNA remnants of viral attacks sometimes get into germ cells (sperm and eggs) and propagate further down the generations. Human genome consists of 3.2 billion base pairs. Only 1.5 % of our genome codes for proteins (genes carry information to make proteins). Interestingly, the rest of the DNA was thought as ´junk´ a couple of decades ago, now we know that they are not junk but carries important instructions for gene production (to drive the protein synthesis from genes). Moreover, now we know that nearly 8.3 % of our genome consists of viral genetic material, contributed by the earlier viral infections. That means a good fraction of our genetic material is due to the viral infections and subsequent integrations of the viral genetic materials- that might have happened millions of years ago to our common ancestors, even before Homo Sapiens emerged. Human genome can be called as a ‘cemetery’ for such retroviral infections, that might have happened to our earlier ancestors.

Some of these viral genes may land on right locations in our DNA genome strand and may get repurposed into some cellular functions (a novel function even). Here is one example how we repurposed a viral protein for a very vital function in our body. A viral gene called ´Env´ which is on the surface of a retrovirus helps them to fuse to the host cells they attach. This process very important for the entry of the virus into the cells. Inside the cells, the viral genetic material will produce more of this Env protein along with other viral proteins, which is directed on the surface of the infected cell. The host cell that expresses this Env protein will fuse with the neighboring cells and form single big fused cells called ´Syncytium´ (see the diagram below). This is a technique employed by the virus to spread easily from one cell to another. In year 2000, a Scientific team in Boston, USA discovered a peculiar gene in the human genome that codes a protein (later they named ´syncytin´) made only by cells in the placenta. Syncytin helps to form a layer of fused cells, which is important for the proper functioning of the placenta. This layer of fused cells forms the outer lining of the umbilical cord and with the placenta, helps in nutrient exchange between mother and fetus. A protein, thus donated to mammals by one of the ancient viral infections, helped us to transform from our egg laying mammal ancestors to placental mammals.

Host cell that expresses Env protein will fuse with the neighboring cells and form single big fused cells called ´Syncytium´. Image from ***https://viralzone.expasy.org/5957?outline=all_by_species***

Syncytin a protein we domesticated from virus, helps to form a layer of fused cells, which is important for the proper functioning of the placenta. This layer of fused cells forms the outer lining of the umbilical cord and with the placenta, helps in nutrient exchange between mother and fetus. Image from ***https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3000028***

Very recently there came some reports on repurposing (exaptation) of parts of a viral gene even in our brain. Retrovirus (mentioned earlier; the family of viruses that includes HIV) uses ´Gag´ gene to produce a protein that forms a protein shell called capsid to protect the genetic material of the virus. A protein in our brain called Arc (this protein is known to play an important role in memory formation) contains parts of the Gag which helps Arc to form functional capsid like structures, that may be vital for its function and thus memory process. This is another example of how a viral gene (donated by an ancient viral infection) is repurposed for functions like neuronal communication.

Viral vectors as gene delivery vehicles

Recent years have witnessed a tremendous development of molecular tools for editing the genome, gene regulation and visualising and manipulating cellular functions, allowing research that until recently was considered ‘science fiction’. There exist several ways such as chemical methods, physical systems and viral vectors, to introduce such tools to cells in a laboratory for research purposes or in the clinic to cure diseases.Viral vectors stand out as the most efficient foreign gene delivery system, especially for applications inside living organism (in vivo). Viruses that have evolved through millions of years can invade all kinds of cells (from tiny bacteria to human cells), hijack the host cell’s machinery to multiply themselves and spread. One can alter the viral genetic material to remove viral proteins essential for its replication and introduce foreign gene(s) of interest, instead. Non-replicative ´gutless´ viral vectors thus produced have the innate ability to target, enter inside the host cells and produce proteins of interest instead of viral proteins. Such viral vectors are highly efficient for difficult-to-transfect (transfection = scientific term for introducing foreign gene into host cells) cells, for studying biological mechanisms in tissue slices or in vivo systems, and recently for introducing genes in human patients in clinics. Viral vectors are clinically approved to deliver functional copies of faulty genes to fix human genetic disorders. No need to mention the tremendous application of viral vectors in research, even cell-type specificity can be achieved to an extent by using a combinatorial approach of conditional viral vectors with the ever-expanding list of transgenics models.

Common viral vectors

Various viruses evolved specific features, which we can exploit for particular research questions. Moloney murine leukaemia virus, for example, is only expressed in dividing cells, and hence suitable for studying cellular mechanisms of dividing cells alongside neighbouring mature cells in intact tissue (thereby a valuable tool for adult neurogenesis research). Rabies virus jumps across the synapses between neurons, but only in the backward direction (retrograde) and is therefore efficient for tracing neuronal connections in the intact brain (mapping connections in the brain). Adeno-associated viruses are the most popular vectors. They are easy to synthesize, do not integrate into the host cell’s genome and can target many types of cells. Lenti virus is useful for long-term gene expression both in vitro and in vivo. Some viruses evolved coat proteins to target only particular kinds of cells, termed innate tropism. By leveraging the innate tropism of a virus, or using cell-specific regulatory elements (we can design viral vectors to target specific type cells in an organ), one can target and introduce therapeutic molecules to specific cells and thereby avoid side-effects in neighbouring cells, a major concern in modern medicine.

Production of viral vectors in a lab

Viral vectors are prepared using mammalian cells as ´production factories´ in a sterile, confined environment with strict safety regulations (biosafety level 2 is enough for gutless viral vectors). Vital replication genes from a viral genome are replaced with foreign genes of interests using molecular biology techniques. Along with the genes/instructions to make our desired protein, additional essential genes are provided separately (in-trans) to cells that make the viral vectors. The gutless viral vectors thus created are infectious, produce exclusively the foreign gene in host cells and since it lacks the replication genes in its genome, they cannot make active, harmful virus particles in any infected cells. Recombinant viral vectors hence work as safe ‘cargo vans’ for delivering our cargo genes. These delivery vehicles with foreign cargo genes are further purified and administered to specific regions (e.g. retinal injections to fix blindness). Please see the simplified diagram below. Cargo genes will be expressed in infected cells and viral vectors being non-replicative, it cannot further infect neighbouring cells like a disease-causing natural virus.

Make a cocktail of all the genetic instructions (DNA) to make virus (viral vector) and introduce into cells in a culture plate. The cells will act as a virus factory. After several days purify the viral vector which can be used for downstream applications.

The top image is a culture plate with cells producing viral vector with the code of a red fluorescent protein . Upon shining those cells with a green light you can see those virus producing cells as fiery red spots. The below image is the microscopic view of the cells (red spots) producing virus.

Clinical applications (gene therapy)

We can alter the viral genomes by taking away the viral proteins and replacing them with the genes of our interest and use the ability of viruses to infect cells and produce the molecules of our interest instead of the viral proteins. Such genetically modified viruses (viral vectors) are used as delivery vehicles/vectors for genes, as research tools and recently to fix genetic diseases. It is not science-fiction anymore, such gene therapies are already in clinics. Here are a couple of examples how viral vectors are used for curing genetic diseases. Recently, FDA approved one-time viral based gene therapy for curing the genetic disorder causing Retinal dystrophy that lead to blindness. Some unfortunate kids are born with mutations in both copies of the RPE65gene, they can experience sight loss at the early age which will progress to total blindness. A functional copy of the RPE65 gene provided by Luxturna can restore vision in children and adults.

Another viral based gene therapy restores functioning of immune systems in young children with a severe disorder caused by mutations in a gene that is crucial for immune-system development, causing severe combined immunodeficiency disorder (SCID-X1). The disease is called ‘bubble-boy’ disease because of the plastic enclosures in which they live, to protect affected children from possible infection. For them, even a common cold can be fatal. The gene-therapy treatment introduces the functional copy of gene IL2RGrestoring the proper immune system. More recently a tool for fixing DNA errors (CRISPR) is entering into the clinics to fix the genetic disorders.

Viral vector-based vaccines (a COVID-19 story)

Beijing Institute of Biotechnology (China) and Oxford University (UK) are conducting clinical trials for vector based COVID-19 vaccine. The Jenner Institute’s (University of Oxford) Vector based COVID-19 vaccine a promising vaccine candidate for COVID-19. It is called ´ChAdOx1 nCoV19´ which is the abbreviation of Chimpanzee Adenovirus Oxford1 novel CoronaVirus 2019). They use another crippled Adenovirus, that cannot replicate in host cells, to deliver the code for spike protein of SARS CoV2 and subsequently to elicit the immune response. The genetically modified Adenovirus will infect the host cells but cannot replicate itself. Upon infection, it faithfully expresses the cargo it carries (here the spike protein of SARS CoV2). This foreign protein will evoke the defense system and produce the appropriate immune mechanisms in vaccinated host. Another example for viral vector-based vaccine is one with brand name ´Ervebo´, that uses another genetically modified virus to produce an important protein of ebola virus as antigen. More such vaccines are in the pipeline using these platforms.

Viral vectors, the future technology

There is one thing that is in common, that made all these applications possible-the usage of viral vectors. In all these cases viral vectors are used as delivery vehicles for introducing the genes. Genetically modified gutless Adeno virus, Adeno-associated virus (various serotypes), Moloney murine leukaemia virus, Lenti virus and Rabies virus are common ‘delivery vehicles’ in research nowadays, each having distinct characteristics making them suitable for different applications. Viral vectors became an inevitable cutting-edge tool for life science research, we deploy them to manipulate mechanisms in cells in a culture plate to behaviours of intact animal. They are used as vaccines against deadly diseases. ´Ervebo´ (brand name), uses another genetically modified virus to introduce an Ebola virus protein as ´antigen´ into a host. In clinics, we already started using viral vectors carrying the right copies of the faulty genes, to fix genetic disorders (gene therapy). One of the main drawbacks of several lifesaving medicines is their ´side-effects´- while it does the ´right job´ at the right place besides, it can create complications at the wrong places. In future, viral vectors may be able to deliver therapeutic molecules to specific regions in an organ. Researchers (including us) had already shown in lab animals that we can target specific cells even in a complex organ like brain (like accurately addressing someone and delivering a package in a huge crowd). Active research is happening to polish these valuable tools further both for research and clinical purposes. In recent future, viral vector mediated gene therapy will be playing a vital role in clinics.

Visit our webpage (www.ntnu.edu/kavli/viral-vector-core).

Video of this blog: Viral vector carrying gene works in a cell