Exploring Viral Vectors For Targeted Gene Therapy

Over time, gene therapy has evolved into a promising treatment option for a number of diseases, including cancers, rare inherited disorders and certain infectious diseases. In fact, gene therapies are also being developed for clinical conditions, which currently have no available treatment option. This can be attributed to the fact that gene therapies and genetically modified therapies involve the introduction of a therapeutic transgene / DNA (gene of interest) into living systems, such as a patient’s body. It is worth highlighting that the process of transgene / DNA delivery into living systems requires the use of a variety of vectors. Currently available gene delivery methods may be broadly classified into viral and non-viral categories.

Viral ­and Non-Viral Methods of Gene Transfer

Vectors may be based on viruses or other molecular methods that enable gene delivery. It is worth mentioning that non-viral vectors offer several advantages, including low immunogenicity and a large packaging capacity. However, they are usually less efficient than viral vectors. Additionally, they require certain viral characteristics, specifically related to receptor mediated uptake and nuclear translocation of DNA, in order to improve non-viral gene transfer.

Viral Vectors for Genetically Modified Therapies

Advances in the field of human genetics have enabled the identification of various Mendelian disorders. Additionally, the insights generated from the Human Genome Project have led to a better understanding of genes and their role in disease initiation and propagation, thereby, accelerating drug development research, using DNA as a therapeutic molecule. However, this field is still niche and gradually evolving in the wake of ongoing technological advancements, such as discovery of appropriate vectors, better understanding of human immunology, and development of practical approaches to select clinical targets. Early initiatives in this domain reported that mammalian viruses are an efficient tool for gene delivery, which also have the potential to be used (directly or indirectly) for the treatment of several genetic disorders.

Despite certain setbacks, which were reported in other early studies involving retroviral vectors, there were two noteworthy trials that demonstrated the successful implementation of viral vector-mediated therapeutics. These studies were conducted in patients suffering from X-linked severe combined immunodeficiency (X-SCID) (2000) and ADA-SCID (2002). It is worth mentioning that, in both trials, treated patients reported successful long-term reconstitution of immune functions in the absence of enzyme replacement therapy. Although there were certain genotoxicity-related adverse events reported in the X-SCID trial, the clinical outcomes observed in both trials markedly outperformed the standard of care therapy used. This offered the necessary evidence to support the potential of gene therapies, establishing the foundation for future improvements. It is also worth mentioning that these studies highlighted the need for gene delivery vehicles that are both safe and efficient. Viral vector manufacturing market is anticipated to grow at a CAGR of around 14%, till 2035, according to Roots Analysis.

Types of Viral Vectors

It is a well-established fact that viruses are extremely efficient in delivering genetic material into a specific target cell, whilst managing to evade the host’s immune system by using the host’s cellular machinery to synthesize various structural and non-structural proteins, which later assemble into functional viruses capable of repeating the process in other target cells. These properties make them highly attractive as gene delivery vectors. Using viruses as vectors involves the manipulation of viral genome; essentially all virulence genes are removed (to prevent viral infection) and replaced with a functional copy of a therapeutic gene(s), along with all the necessary regulatory sequences that control its expression. These modified viruses are able to carry specific target cells with high efficiency. As indicated earlier, such a method of gene delivery is called transduction; likewise, a cell modified by a virus / viral vector is said to have been transduced.

1. Adeno-associated Viral Vectors

Adeno-associated virus (AAV) is a small virus of the Parvoviridae family that has a single stranded DNA genome. It is capable of infecting a broad range of host cells, including both dividing and non-dividing cells. It is a non-pathogenic virus that does not generate an immune response in most patients.

The AAV genome comprises of inverted terminal repeats (ITRs) at both ends of the DNA strand and two open reading frames (ORFs), namely rep and cap. Each ITR sequence consists of 145 bases that have the ability to form a hairpin structure. These sequences are required for the primase-independent synthesis of a second DNA strand and the integration of the viral DNA into the host cell genome. The rep genes encode proteins that are required for the AAV life cycle and site-specific integration of the viral genome. Whereas, cap genes encode the capsid proteins, namely VP1, VP2 and VP3.

2. Adenoviral Vectors

Adenoviruses are members of the Adenoviridae family that typically have a double stranded DNA genome. The size of an adenoviral genome is generally around 36 kb, however, such viruses can accommodate cDNA sequences of up to 7.5 kb. When an adenovirus infects a host cell, its genetic material (DNA) is inserted into the host cell, and not into the host’s genome. Instead, it is left free in the nucleus in the form of an extrachromosomal gene segment, which is also known as an episome. The information in this episomal DNA molecule is transcribed and translated in a manner similar to that of any other gene, however, episomes are not passed on to daughter cells post replication.

3. Lentiviral Vectors

Lentiviruses are also RNA viruses that belong to the Retroviridae family. Similar to retroviruses, they are also capable of stably inserting genetic material into the genome of a host cell. However, unlike retroviruses, these vectors can infect non-dividing cells as well. The only cells that lentiviruses cannot gain access to are quiescent cells (those in the G₀ state). This is primarily because cells in the G₀ phase inherently block the reverse transcription step. Examples of lentiviruses include:

• Human immunodeficiency virus (HIV)
• Simian immunodeficiency virus (SIV)
• Feline immunodeficiency virus (FIV)
• Equine infectious anemia virus (EIAV)

4. Retroviral Vectors

Retroviral vectors are RNA viruses that belong to Retroviridae family. Within the host cell, these viruses synthesize double-stranded DNA molecules using RNA as a template; this process is facilitated by an enzyme, known as reverse transcriptase. The newly synthesized DNA can then be integrated into the chromosome of the host cell in a process that is carried out by another enzyme, known as integrase. Stable integration of the DNA synthesized from viral genome serves to modify the host cell, causing it to synthesize viral proteins. It is also worth mentioning that when the modified host cell divides, daughter cells retain copies of the viral genes and continue producing viral proteins.

5. Other Viral Vectors

Other viral vectors are classified under the following categories:

• Alphavirus: Alphaviruses belong to the Togaviridae family of viruses that are capable of infecting both vertebrates and invertebrates. Its genome is a single stranded RNA molecule, which is typically 11 to 12 kb, having a 5’ cap and 3’ poly-A tail. Additionally, the genome contains two ORFs that code for non-structural and structural components. In alphaviruses, the expression of viral proteins and the replication of the viral genome takes place in the cytoplasm of the host cell. It is also worth mentioning that certain retroviral and lentiviral vectors are usually pseudo typed using alphavirus envelope proteins, which facilitate the recognition and infection of a wide range of potential host cells.
• Foamy Virus: Foamy viruses, also known as spumaretro viruses, are known to impart a characteristic foamy appearance to the cytoplasm of the cells they infect, thereby leading to the development of multinuclear syncytia. They are found in several mammals, including cats, cows and captive nonhuman primates, excluding humans. The safety profiles of foamy viruses for clinical purposes has made it a preferred choice as compared to other vectors, such as γ-retroviral vectors. In terms of being used as a gene transfer tool, they offer several unique advantages over other integrating viral vectors, such as gamma-retroviruses and lentiviruses. These include a large packaging capacity (up to 12 kb), broad host and cell-type tropism, and safer integration profile with lower risk of insertional mutagenesis. These vectors can also be used to efficiently transduce quiescent cells; since its genome remains stable (in the form of cDNA) in growth-arrested cells / quiescent cells, it can be integrated into the host genome once the cell exits the G₀
• Herpes Simplex Virus: The herpes simplex virus (HSV) is a double-stranded DNA virus that belongs to the Herpesviridae It is a neurotropic virus that is known to infect humans, which makes it a likely candidate for being used for the transfer of genes to the nervous system. The relatively large genome of the virus enables the insertion of more than one therapeutic gene into a single virion. It is therefore possible to use HSV vectors for the treatment of disorders caused by more than one defective gene. It is worth mentioning that the HSV is able to infect a wide range of cells, including muscle cells, liver cells, pancreatic cells, neurons and lung cells. Typically, these vectors are designed to encode the HSV thymidine kinase enzyme, which makes the virus susceptible to acyclovir mediated inhibition. Acyclovir is a nucleoside analogue that is used to treat HSV infections and in this case, is administered along with the oncolytic virus therapy.
• Sendai Virus: Sendai virus is a non-segmented negative strand RNA virus, which belongs to the Paramyxoviridae It was discovered in 1953 in Japan and was primarily considered for the development of a xenotropic live-attenuated vaccine, owing to its antigenic similarity to the human parainfluenza virus type 1. The virus possesses certain unique characteristics, including a powerful capacity for gene expression, low pathogenicity and broad host range, which makes it a suitable vector candidate for the transfection of various animal cells. Despite the aforementioned benefits, vectors based on this virus are associated with inefficient chromosomal integration (of transgenes). These vectors have been used as a research tool in various life sciences domains, however, its utility as a recombinant viral vector has been identified recently. Owing to its ability to induce mucosal immunity, the vector has been significantly exploited as a vaccine platform. It has also been studied for cancer gene therapy at preclinical stage.
• Simian Virus: Simian virus 40 (SV40) belongs to the Polyomaviridae Typically, these viruses have a circular, double-stranded DNA genome which is 5.2 kb in length. Certain genes that are transcribed at an early phase in the viral life cycle (early genes) include the large T antigen (Tag) and the small tag. Similarly, genes transcribed later during the life cycle of the virus (late genes) include the regulatory protein, agnoprotein and three structural proteins (namely VP1, VP2 and VP3). The Tag gene confers immunogenic properties to the recombinant SV40 viral vector; hence, it is deleted while developing SV40 vectors. The deletion of all the structural proteins, except the major capsid protein VP1, serves to reduce the overall size of the viral genome. The final vector genome is typically made up of the origin of replication and the encapsidation sequence, offering enough space for the incorporation of a transgene.
• Vaccinia Virus: The Vaccinia virus belongs to the Poxviridae family; it is comprised of a linear, double-stranded DNA genome, which is approximately 190 kb in length and codes for close to 250 genes. It has the capability to replicate its genetic material in the cytoplasm of the host. Based on the composition of its outer membranes, the vaccinia virus can be divided into four types, which include intracellular mature virion, intracellular enveloped virion, cell associated enveloped virion and extracellular enveloped virion. This virus is popular owing to its use in the development of the vaccine that enabled the eradication of smallpox. With a packaging capacity of up to 25 kb of foreign DNA, the vaccinia virus can be efficiently used for the delivery of large gene sequences.