Clinical Application of Gene Therapy in Veterinary Medicine

 

Clinical Application of Gene Therapy in Veterinary Medicine

Gene therapy is a medical approach that treats or prevents disease by correcting the underlying genetic problem. Gene therapy techniques allow doctors to treat a disorder by altering a person’s genetic makeup instead of using drugs or surgery. Originally, the term gene therapy referred to proposed treatments of genetic disorders by replacing a defective gene with its normal counterpart. Currently, it includes all treatments in which there is an introduction of genetic material into body cells to treat a variety of diseases.

The earliest method of gene therapy, often called gene transfer or gene addition, was developed to:

• Introduce a new gene into cells to help fight a disease.
• Introduce a non-faulty copy of a gene to stand in for the altered copy causing disease.

Later studies led to advances in gene therapy techniques. A newer technique, called genome editing (an example of which is CRISPR-Cas9), uses a different approach to correct genetic differences. Instead of introducing new genetic material into cells, genome editing introduces molecular tools to change the existing DNA in the cell. Genome editing is being studied to:

• Fix a genetic alteration underlying a disorder, so the gene can function properly.
• Turn on a gene to help fight a disease.
• Turn off a gene that is functioning improperly.
• Remove a piece of DNA that is impairing gene function and causing disease.

Gene therapies are being used to treat a small number of diseases, including an eye disorder called Leber congenital amaurosis and a muscle disorder called spinal muscular atrophy. Many more gene therapies are undergoing research to make sure that they will be safe and effective. Genome editing is a promising technique also under study that doctors hope to use soon to treat disorders in people.

Gene therapy offers great opportunities to treat or cure disease and alleviate human as well as animal suffering. The majority of the infants that were afflicted by the devastating disease and that were subsequently treated by gene therapy can now essentially lead normal lives. Now this advanced technology is showing great achievement in the treatment of various type of genetic or non-genetic diseases in human or animals. Most importantly, these successes offer new therapeutic options for patients who are currently untreatable. Convincing evidence continues to emerge that gene therapy is effective in patients suffering from other hereditary diseases besides the congenital immune deficiencies (e.g. β- thalassemia, hemophilia and epidermolysis bullosa) but also from more common disorders like cancer, neurodegenerative or cardiovascular disorders. Even patients (including animals) suffering from an inborn genetic disease that is not life-threatening but causes blindness can finally start to see by gene therapy. These few selected recent examples of clinical advances in gene therapy clearly indicate that the momentum in this new approach of treatment is building up. In the absence of effective drugs or alternative therapies, gene therapy technology may play a significant role and also best hope for the many patients suffering from various diseases. In the present scenario when there are global economic challenges, it is even more important than ever to find sustainable solutions to treat diseases of high unmet medical needs. The potential for a one-time curative treatment by gene therapy permanently solve continuous therapeutic interventions for the treatment of chronic diseases. So to reduce the economic burden it is absolutely essential to further consolidate and exert sustainable effort towards the gene therapy.

Objective

• To compensate for a missing protein in a genetic disease.

• To inhibit the production of an undesirable protein (by generating an antisense mRNA).

• To express a trophic factor or an anti-inflammatory cytokine.

• To introduce a suicidal gene for the treatment of cancer.

 

Approaches for gene therapy ———

There are two approaches to achieve gene therapy:

1) Somatic gene therapy– It involves the insertion of a functional and expressible gene into a target somatic cell to correct a genetic disease. It represents the mainstream line of current basic and clinical research where any modifications and effects will not be inherited by the patient’s offspring or later generations. Somatic gene therapy is viewed as a more conservative and safer approach because it affects only the targeted cells in the patient and is not passed on to future generations; however, somatic cell therapy is short­lived because the cells of most tissues ultimately die and are replaced by new cells. In addition, transporting the gene to the target cells or tissue is also problematic. Regardless of these difficulties, however, somatic cell gene therapy is appropriate and acceptable for many disorders .

2) Germline gene therapy- In this approach, functional genes are introduced into germ cells (sperm or egg). Therefore the changes due to therapy would be heritable. Although this approach is highly effective in counteracting genetic and hereditary diseases, but for safety, ethical and technical reasons, germline gene therapy is not being attempted at present. The genetic alterations in somatic cells are not carried to the next generations. Therefore, somatic gene therapy is preferred and extensively studied with an ultimate objective of correcting human diseases.

Types of gene therapy——–

There are two types of gene therapy :- 1. Ex vivo gene therapy :

This technique involves the following stepsa) Isolate cells with genetic defect from a patient

b) Grow the cells in culture

c) Introduce the therapeutic gene to correct gene defect

d) Select the genetically corrected cells and grow

e) Transplant the modified cells to the patient.

The procedure basically involves the use of the patient’s own cells for culture and genetic correction, and then their return back to the patient.

 

In vivo gene therapy: The direct delivery of the therapeutic gene into the target cells of a particular tissue constitutes in vivo gene therapy. Many tissues are the potential candidates for this approach. For example liver, muscle, skin, spleen, lung, brain and blood cells etc.

The success of in vivo gene therapy mostly depends on the following parameters:

• The efficiency of the uptake of the therapeutic gene by the target cells

• Intracellular degradation of the gene and its uptake by nucleus

• The expression capability of the gene.

Techniques of gene transfer (Vectors in gene therapy)——-

The most fundamental requirement for gene therapy to be successful is to effectively deliver the therapeutic gene to the target cell. The carrier particles or molecules used to deliver genes are referred to as vectors. There are different viral and non-viral vectors for gene delivery. The ideal gene delivery vector should be very specific, capable of efficiently delivering one or more genes of the size needed for clinical application and unrecognized by the immune system. Finally, a vector should be able to express the gene for as long as is required.

Gene delivery by viruses

Viruses are one of the most promising vectors currently being used for gene therapy. Viruses are actually genes wrapped in a protein coat. This coat contains special proteins that can bind to the surface of cells. Once bounded they either force their way in or are sucked into the cell itself. Scientists have tried to take advantage of this capability and manipulate the viral genome and replace them with working human gene. Once the transplanted gene is ‘switched on’ in the right location within the cells of an infected person, it can then issue instructions for making specific proteins.

Some of the different types of viruses used as vectors in gene therapy:

 

• Retroviruses————- Retroviruses were the first viruses to be used as vectors in gene therapy experiments. They contain the enzyme reverse transcriptase which can create double- stranded DNA copies of their RNA genomes. These copies of its genome can be integrated into the chromosome of host cell. Although retroviruses have been used in most gene therapy experiments, there are some drawbacks. The main limitations of retroviral vectors are their low efficiency in vivo, immunogenic problems, the inability to transduce the non-dividing cells and the risk of insertion, which could possibly cause oncogene activation or tumour-suppressor gene inactivation .

However, with development of our understanding of the function of nucleases such as zinc finger nucleases in humans, the genes encoding nucleases are incorporated into chromosomes; the expressed nucleases then “edit” the chromosome, disrupting genes causing disease. Treatment of the X- linked severe combined immune deficiency using retroviral vector represent the most successful application of gene therapy till date .

 

• Adenoviruses—————

Adenovirus type 2 and 5 can be utilized for transferring both dividing and non-dividing cells and have low host specificity so can be used for gene delivery into large range of tissues. The vectors based on adenovirus are generally used for therapeutic strategies that require the therapeutic gene to be active for only a short time. However, it is a disadvantage where sustained gene activity is required for many months such as in the treatment of some tumours, neurodegenerative disease and HIV infection. Natural and acute immunologic responses against adenoviruses have made their clinical application limited to a few tissues, such as liver, lung (especially for Cystic Fibrosis), or localized cancer gene therapy (12,13). Although the risk of serious disease following natural adenovirus infection is rare and the viral genome would not integrate into the host genome, gene therapy by adenoviral vectors has caused serious bad side effects and even death of some patients. Recently, in addition to safety of these vectors, several essential genes have been deleted so that viral replication can only occur under control and also most of the viral genome is deleted to obtain sufficient space for transgene particles, this kind of adenoviruses are called “gutless” or “pseudo” adenoviruses.

 

Speculative uses for gene therapy ———

 

Gene doping A number of gene therapies have potential applications to athletic enhancement and the gene therapy technologies might be abused to improve athletic performance. This is known as gene doping.(35) ü Human genetic engineering It has been speculated that genetic engineering could be used to change physical appearance, metabolism and even improve physical capabilities and mental faculties. These speculations have led to ethical concerns and claims, including the belief that every foetus has an inherent right to remain genetically unmodified. Pros and cons of gene therapy The positive aspect of gene therapy is apparent. Gene therapy is a “medicine” for the future since it can wipe out genetic diseases before they can begin and eliminate suffering for future. However, no therapy is without some associated risks. Some of the problems associated with gene therapy are: • Immune response- Genes injected with a virus may trigger an immune response in the body. • Multigene disorders- Disorders arising from single gene mutation are best candidates for gene therapy. Unfortunately, some of the most commonly occurring diseases, such as heart disease, hypertension, Alzheimer’s disease, arthritis and diabetes are multigene disorders. • Insertional mutagenesis- If the DNA is integrated in wrong place in the genome, for example in a tumour suppressor gene, it could induce a tumour. • Problems with viral vectors- The viral vector may recover its ability to cause disease. • Short-lived nature There are several ethical and legal issues associated with gene therapy. A review board, the Recombinant Advisory Committee (RAC) has been developed to address these concerns. The consequences of gene therapy are many. The first issue targets putting human fate in our own hands. Some people are concerned that gene therapy could be used for any genetically linked trait such as eternal appearance, personality or physical enhancement. Another great concern is religion. Some consider it sinful to manipulate DNA. If religion is a factor then somatic cell therapy should be applied which allows the next generation to make their own decision. In addition to ethical issues, one of the major concerns is the cost of gene therapy. However, scientists are optimistic that the cost will be much cheaper in future. The last but not the least is the risk of the procedure. Since gene therapy is still in its developmental stage, finding the precise location of the gene and replacing with a normal one is definitely a challenge. But it is true that with the invention of new and advanced techniques, researchers will soon be able to achieve a great success in this applied modern science.

 

History of Gene Therapy

 

Genetic engineering that was first presented at the Sixth International Congress of Genetics held in 1932, the concept of genetic correction arose after Avery, MacLeod and McCarty in 1944 suggested that “genes could be transferred within nucleic acids” . Clyde E. Keeler in 1947 was probably the first to use the term gene therapy, although the process he was describing (the correction of gene-based deviations in plants and animals) was not envisaged as an effective therapeutic technique to treat genetic diseases in man . Lederberg, 1963 – The first real contribution to the field is attributed to Nobel Prize winner Joshua Lederberg, a pioneer in bacterial genetics and plasmid biology, and a visionary in gene therapy.

Obstacle in the Development of Gene Therapy

 Short Life of Treatment

The therapeutic genetic material introduced into target cells must remain functional. Naked DNA or certain viruses (e.g. AAVs) may remain episomal and allow sustained expression in stable tissues (e.g. neurons or skeletal muscles).

Toxicity and Inflammatory Responses

In the OTCD trial, the patient J. Gelsinger died from fulminant hepatitis four days after beginning treatment with an adenovirus vector. Since then, work using adenovirus vectors has focused on genetically crippled versions of the virus, safer production standards and clinical protocols.

 

Insertional Mutagenesis

This has occurred in clinical trials for X-linked severe combined immunodeficiency (SCID-X1) patients, in which hematopoietic stem cells were transduced with the gamma-chain interleukin 2 (C IL2) receptor gene using a retrovirus, and this led to the development of T-cell leukemia in 5 out of 20 patients. All but one of these children responded well to conventional antileukemia treatment.

 

Significance of Recent Progress of Gene Therapy

Deeper Understanding of the Biology of the Transduced Cells

For instance, for treatment of SCID patients, using myeloablative preparative treatment which was not included in the early trials.

 Improvements in Our Understanding of the Vectors

The issue of insertional mutagenesis has been addressed by replacing retroviruses by lent viral vectors (first evaluated in 2006 in HIV using genetically modified CD4 T-cells, and now in other immunodeficiencies), or by including certain sequences such as the globin locus-control region to direct to specific chromosomal site. Other forms of safer genetic engineering include gene targeting and knocking-out specific genes via engineered nucleases.

Improved Delivery Procedures

The injection of naked DNA is a perfect example of this issue. Clinical trials of intramuscular injection of a naked DNA plasmid have occurred with some success; however, expression has been very low in comparison to other methods of transfection research efforts focusing on improving the efficiency of naked DNA uptake have yielded several novel methods, such as electroporation, sonoporation, and the use of a “gene gun” (ballistic gold, DNA-coated gold particles). The advanced current indications are DNA vaccines, and angiogenic cardiovascular applications, as they involve only short-term expression and local delivery.

How does Gene Therapy Works?

There are several ways through which gene therapy works.

• Replace a mutated gene with a healthy version of that gene.
• Introduce a new functioning gene to fight disease.
• Inactivate a faulty gene that is causing disease.

This gene therapy diagram shows that first, the defective genes are spotted. Then, medical experts use healthy genes to replace faulty ones. Finally, the new gene restores the functionality of the existing cells. Some portions of DNA containing useful proteins enter the cells through the vectors. Inside the cell, DNA/genes start making useful proteins. After some time, the damaged cells heal and remove the source of the disease.

The next segment focuses on the varied application of gene therapy. 

Different Kinds of Gene Therapy

Primarily, there are two types of gene therapy. 

• Somatic Gene Therapy 

The human body mainly consists of somatic or stem cells. This process uses healthy genes to replace damaged ones. The therapy targets the defective cells of an individual who is suffering from a disease. Somatic cells are mainly non-reproductive. That means the effects of this therapy will not transfer to the future generation. Hence, it is considered to be one of the safest applications of gene therapy.

• Germline Gene Therapy 

This therapy targets the germ cells of the body that produce eggs or sperms. Germline gene therapy process includes the infusion of functional DNA into cells. However, the effect of this therapy can affect future generations. Therefore, the usage of this therapy is restricted in many places. For example, the European Union does not allow this process.

Gene Therapy Application

With time, the popularity of this therapy is increasing. The application of gene therapy includes the following:

• Effectively cures several genetic disorders.
• Treats diseases like brain tumours, Alzheimer’s, Parkinson’s, Haemophilia, and several others.
• Useful for the diseases that traditional medicine cannot cure.
• Solely destroys disease-causing cells without affecting other cells.
• Can be used on individuals, as well as embryos.

However, this treatment has some temporal or permanent side effects too.

Challenges of Gene Therapy 

• The new genes have to reach the right place.
• On reaching the exact location, this gene has to start becoming functional.
• The genes can cause harm if they reach the wrong cells.
• Sometimes targeted cells stop the new genes from entering. The immune system of a body often also tries to kill the inserted gene.
• It has to make sure that the new cells are affecting the functions of other cells.

In short, gene therapy can be an excellent treatment option if used properly.

Clinical Application of Gene Therapy

 

1. Oncolytic Adenoviruses for Cancer Gene Therapy –———-Relatively novel and promising therapeutic platform is virotherapy with oncolytic adenoviruses. Over the last two decades several engineered viral mutants have been evaluated in clinical trials targeting various tumor types and were demonstrated to be safe, with some efficacy. This approach has been applied to numerous viral species, including adenovirus, measles, herpes and poxviruses, to name a few. However, most of the work has focused on adenoviral vectors, especially serotype 5, because of the ease of genetically engineering its small, linear and well characterized 36 kb genome. Additionally, adenoviral mutants can be engineered to not only lyse cancer cells but also express therapeutic transgenes to promote elimination of tumors. Another advantage of adenovirus is the ability to infect both proliferating and nonproliferating tumor cells, an important consideration in many solid tumors with only sub-populations of cells that are actively dividing (e.g. prostate cancer).

 

Developmental Process

There are several approaches to engineering replication-selective, adenoviral mutants:

deletion of viral genes that are essential for the viral life-cycle to proceed in normal cells but are functionally complemented by the altered gene expression in cancer cells insertion of tumor-/ tissue-specific promoters to control expression of early viral genes that drive replication modification of viral tropism to specifically target tumor antigens and infect cancer cells only insertion of microRNA (miRNA) target sequences to suppress expression in normal cells and combining any or all of the approaches above, with or without expression of therapeutic genes e.g. small RNAs, antiangiogenesis factors.

 

1. Gene Therapy of the Beta (β) Hemoglobinopathies

Patients of beta hemoglobinopathies who inherit two different mutations may be severely affected whereas heterozygote carriers are generally asymptomatic. In some populations where malaria is endemic, the prevalence of Hb defects can be as high as 40%. Most affected patients in developing countries die before the age of five years, whereas most of the affected children born in high-income countries survive but live with a chronic and severe disorder. β- hemoglobin disorders fall into two large groups of hemoglobin (Hb) mutations: structural variants, in which amino acid changes produce abnormal Hb such as Hb S, E, D or C and βthalassemia’s, in which β-globin chain production is low or nonexistent. Patients who inherit two different mutations may be severely affected whereas heterozygote carriers are generally asymptomatic. In some populations, where malaria is endemic, the prevalence of Hb defects can be as high as 40% .

 

Beta-Thalassemias

 

There is very a wide range of clinical severity, from the severe transfusion- dependent thalassemia major to the highly variable no transfusiondependent thalassemia intermedia. They can be caused by a number of mutations at the globin locus resulting in either no globin production or reduced levels of synthesis that lead to imbalanced ɑ:non- aglobin chain ratios. Excess a-chain damages the cell membrane, leading to apoptosis and anemia. In β -thalassemia, the aim is to achieve cellular levels of β-globin expression that are sufficient to bind much of the unpaired a-chains, and this level varies whether there is complete absence of endogenous β-globin expression-thalassemia. Regular transfusions and a strict iron chelation program are warranted to assure patient survival. Without treatment, patients succumb within two years.

Sickle Cell Disease

James Herrick in 1910 reported the first case of a patient with a typical elongated red blood cells (RBCs) and these findings led to the description of sickle cell anemia in 1922. It was due to nucleotide change from GAG to GTG. This valine for glutamic acid substitution at codon 6 is responsible for HbS polymerization, which is theprimary molecular event leading to RBC defects, sickling, hemolysis, increased blood viscosity, vaso- occlusion, painful crises, strokes and multi-organ damage.

 

Gene Therapy of Retina

1. Strategies for Dominant Mutations in the case of dominant inherited retinal degeneration, the normal protein are not expressed as per desire of the body and one must also suppress expression of the mutant protein to obtain a therapeutic effect. The proposed gene therapy strategies are to remove the endogenous gene expression using molecular tools and express a normal gene copy artificially. b. Neuroprotection A large number of trophic factors, e.g. ciliary neurotrophic factor (CNTF), glial-cell-line-derived neurotrophic factor (GDNF) and rod-derived cone viability factor (RdCVF), have demonstrated their ability to protect retinal neurons. So gene therapy represents a priori, an ideal solution for delivery of these factors into the eye. c. Ocular Neovascularization is an essential symptom in the onset of blindness for eye disease representing a crucial public health issue such as wet-AMD or diabetic retinopathy.Vascular endothelial growth factor (VEGF) has been identified as a major player in the onset of neovascularization. Consequently, a new class of pharmacological agents, designed to block VEGF action, has been put into use, dramatically improving the management of wet-AMD patients. d. Optogenetics is a new emerging area of neuroscience based on targeted expression in neurons of bacterial ion channels whose opening is triggered by light and leads to depolarization of the neuronal cell.These tools have led to significant progress in our understanding of the neural circuits of the CNS.

Gene Therapy for Hemophilia Aand B

Hemophilia A and B are congenital bleeding disorders caused by a deficiency of functional clotting factor, FVIII and FIX, respectively. Hemophilia results in an X-linked bleeding diathesis caused by a mutation in the corresponding clotting factor genes. It affects an estimated 400,000 individuals worldwide (according to the World Federation of Hemophilia). Hemophilia A affects nearly 80–85% of the patients, whereas the remaining 15% are afflicted by hemophilia B. As FVIII and FIX are essential cofactors in the blood coagulation cascade, patients suffer from recurrent bleeding and chronic damage to soft tissues, joints and muscles. This progresses towards chronic synovitis, crippling arthropathy and physical disability. The bleeding could also be fatal in the case of intracranial hemorrhage.

Hemophilia as ATarget of Gene Therapy

The introduction of a functional FVIII or FIX gene copy into the target cells via gene therapy may provide a cure and eliminate the need for repeated clotting factor infusions.

Gene Therapy Products

• Plasmid DNA – Therapeutic genes may be genetically built into circular DNA molecules and delivered to human cells.
• Viral Vectors – Several gene therapy products are generated from viruses because viruses have the inherent capacity to transport genetic material into cells. Once viruses have been engineered to lose their potential to cause infectious illness, they can be employed as vectors (vehicles) to deliver therapeutic genes into human cells.
• Bacterial Vectors – Bacteria can be altered to avoid producing infectious illness and then utilised as vectors (vehicles) to deliver therapeutic genes into human cells.
• Human gene Editing Technology – Gene editing aims to either disrupt dangerous genes or fix mutated genes.
• Patient-derived Cellular Gene Therapy Products – Cells are extracted from the patient, genetically changed (typically with the use of a viral vector), and then returned to the patient.

Disadvantages of Gene Therapy

• DNA Mutations – The new gene might be placed in the incorrect place in the DNA, resulting in dangerous DNA alterations or even cancer.
• Immune Response – The body’s immune system may recognize the newly added viruses as invaders and attack them, resulting in inflammation, toxicity, and, in severe cases, organ failure.
• Viral Spread – Because viruses may impact several types of cells, it is likely that the viral vectors will infect cells other than those with altered or absent genes. If this occurs, healthy cells may be destroyed, resulting in sickness or disease, including cancer.
• Risk to Offspring – The altered DNA might have an impact on reproductive cells, such as egg cells in women and sperm cells in men. This might lead to genetic alterations in children born after the therapy.
• Reversion of the virus to its Original Form – When viruses are introduced into the body, they may regain their potential to cause disease.
• High cost
• Potential for short-term efficacy

Which Diseases does Gene Therapy Treat?

Several gene therapies have been authorised by the FDA. CAR T-cell therapy is one of the first, and it’s exclusively for children and young people with B-cell acute lymphoblastic leukaemia, who have failed to respond to prior therapies. In Europe, a treatment for lipoprotein lipase deficiency, a condition in which a person is unable to break down fat molecules, was authorised as the first gene therapy in 2012.

Another treatment for severe combined immune deficiency (also known as “bubble boy” syndrome) might be available in Europe very soon. Experiments have shown promising outcomes for a variety of additional ailments, that includes – Haemophilia. Some cause blindness, immune deficiencies, muscular dystrophy. Many more clinical studies are now underway, many of which are for uncommon diseases.

EDITED & COMPILED BY-TEAM SAVERA