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Below are some of the most common questions raised regarding gene therapy. The material is provided by the American Society of Gene & Cell Therapy and can be found at

What is gene therapy?

In the broadest sense, gene therapy is the introduction, removal, or change in the content of a person’s genetic code with the goal of treating or curing a disease. The transferred genetic material changes how a single protein or group of proteins is produced by the cell. Gene therapy can be used to reduce levels of a disease-causing version of a protein, increase production of disease-fighting proteins, or to produce new/modified proteins.

Yes! At the time of writing, lists more than 1000 different types of gene therapy in clinical trials.

Gene addition involves inserting a new copy of a gene into the target cells to produce more of a protein. Most often, a modified virus such as adeno-associated virus (AAV) is used to carry the gene into the cells. Therapies based on gene addition are being developed to treat many diseases.

Gene correction can be achieved by modifying part of a gene using recently-developed gene editing technology (e.g. CRISPR/cas9, TALEN or ZFN) to remove repeated or faulty elements of a gene, or to replace a damaged or dysfunctional region of DNA. The goal of gene correction is to produce a protein that functions in a normal manner instead of in a way that contributes to disease.

Gene silencing prevents the production of a specific protein by targeting messenger RNA (mRNA; an intermediate required for protein expression from a gene) for degradation so that no protein is produced.

Gene reprogramming involves adding one or more genes to cells of a specific type to change the characteristics of those cells. This technique is particularly powerful in tissues where multiple cell types exist and the disease is caused by dysfunction in one type of cells.

Cell elimination uses strategies that are typically used to destroy malignant (cancerous) tumor cells, but can also be used to target overgrowth of benign (non-cancerous) tumor cells. Tumor cells can be eliminated via the introduction of “suicide genes,” which enter the tumor cells and release a prodrug that induces cell death in those cells. Viruses can be engineered to have an affinity for tumor cells.

How does gene therapy work?

Put simply, gene therapy works by changing the genetic information of a population of cells in a way that alleviates or combats the cause or symptoms of a disease.

Risks of any medical treatment depend on the exact composition of the therapeutic agent and its route of administration. Different types of administration, whether intravenous, intradermal or surgical, have inherent risks.

Viral vectors and oncolytic viruses are designed to reduce the risk of adverse effects, and each viral vector is rigorously tested in cells and animals before it is considered for human use. The viral vectors used in human trials are prepared under strict guidelines to ensure purity and integrity.

How are genes delivered?

Scientists and clinicians use the following four methods to carry genetic material into the targeted cells.

Simple strands of naked DNA or RNA can be pushed into cells using high voltage electroporation. This is a common technique in the lab. Naked DNA or RNA may also be taken up by target cells using a normal cellular process called endocytosis after addition to the medium surrounding the cells. Finally, sheer mechanical force can be utilized to introduce genetic material with an instrument called a “gene gun.”

Genetic material can be packaged into artificially-created liposomes (sacs of fluid surrounded by a fatty membrane) that are more easily taken up into cells than naked DNA/RNA. Different types of liposomes are being developed to preferentially bind to specific tissues. Recent work has utilized a subtype of membrane vesicles that are endogenously produced and released by cells (extracellular vesicles or “exosomes”) to carry small sequences of RNA into specific tissues.

Viruses have an innate ability to invade cells. The symptoms of a cold are triggered by a cold virus entering the cells of the upper respiratory tract and hijacking the cell’s machinery to manufacture more virus. Viral vectors for gene therapy are modified to utilize the ability of viruses to enter cells after disabling the capability of the virus to divide. Different types of viruses have been engineered to function as gene therapy vectors. In the case of adeno-associated virus (AAV) and retrovirus/lentivirus vectors, the gene(s) of interest and control signals replace all or most of the essential viral genes in the vector so the viral vector does not replicate. For oncolytic viruses, such as adenovirus and herpes simplex virus, fewer viral genes are replaced and the virus is still able to replicate in a restricted number of cell types. Different types of viral vector preferentially enter a subset of different tissues, express genes at different levels, and interact with the immune system differently.

Cells are collected from the patient or matched donor and then purified and expanded in vitro. Scientists and clinicians then deliver the gene to the cells using one of the three methods described above. Those cells that express the therapeutic gene are then re-administered to the patient.

How are viruses used in gene therapy?

Viruses are used in gene therapy as gene delivery vectors and as oncolytic viruses:

Viruses as gene delivery vectors. Modified viruses are used as carriers in gene therapy. These viral vectors protect the new gene from enzymes in the blood that can degrade it, and deliver it to the relevant cells. Viral vectors efficiently coerce the cells to take up the new gene, uncoat the gene from the virus particle, and transport it, usually to the cell nucleus. The transduced cells begin using the new gene to perform its function, such as synthesis of a new protein. Viral vectors are genetically engineered so that most of their essential genes are missing, which prevents uncontrolled replication of the virus and makes room for insertion of the gene to be delivered.

Many different viral vectors are being developed because the requirements of gene therapy agents for specific diseases vary depending on the affected tissue, the level of gene expression, and the required duration of expression. Scientists examine the following characteristics while choosing or developing an appropriate viral vector: (i) size of DNA or gene that can be packaged, (ii) efficiency of uptake by the desired cells for therapy, (iii) duration of gene expression, (iv) effect on immune response, (v) ease of manufacturing, (vi) ease of integration into the cell’s DNA or ability to exist as a stable DNA element in the cell nucleus without genomic integration, and (vii) chance that the patients have previously been exposed to the virus and thus might have antibodies against it which would reduce its efficiency of gene delivery.

Oncolytic Viruses

Oncolytic viruses are engineered to replicate only or predominantly in cancer cells and not in normal human cells. Once oncolytic viruses replicate in cancer cells they cause the cancer cells to burst, releasing more oncolytic viruses to infect surrounding cancer cells.

What are some of the challenges gene and cell therapists face?

The challenges of gene and cell therapists can be divided into three broad categories based on disease, development of therapy, and funding.

Disease symptoms of most genetic diseases, such as AHC, are caused by distinct mutations in single genes. Note that there are many susceptible genes and additional mutations yet to be discovered. Gene replacement therapy for single gene defects is the most conceptually straightforward. However, even then, the gene therapy agent may not equally reduce symptoms in patients with the same disease caused by different mutations. Even the same mutation can be associated with different degrees of disease severity. Gene therapists often screen their patients to determine the type of mutation causing the disease before enrollment into a clinical trial.

Disease models in animals do not completely mimic human diseases, and viral vectors may infect various species differently. The testing of vectors in animal models often resembles the responses obtained in humans, but the larger size of humans in comparison to rodents presents additional challenges in the efficiency of delivery and penetration of tissue. Gene therapy, cell therapy, and oligonucleotide-based therapy agents are often tested in larger animal models, including rabbit, dog, pig, and nonhuman primate models. Testing human cell therapy in animal models is complicated by immune rejections. Furthermore, humans are a very heterogeneous population. Their immune responses to the vectors, altered cells, or cell therapy products may differ or be similar to results obtained in animal models.

Scientific challenges include the development of gene therapy agents that express the gene in the relevant tissue at the appropriate level for the desired duration of time. After the delivery modalities are determined, identification and engineering of a promoter and control elements (on/off switch and dimmer switch) that will produce the appropriate amount of protein in the target cell can be combined with the relevant gene.

Furthermore, the immune system’s response needs to be considered based on the type of gene or cell therapy being undertaken. For example, to treat genetic diseases like hemophilia and cystic fibrosis, the goal is for the therapeutic protein to be accepted as an addition to the patient’s immune system.

If the new gene is inserted into the patient’s cellular DNA, the intrinsic sequences surrounding the new gene can affect its expression and vice versa. Scientists are now examining short DNA segments that may insulate the new gene from surrounding control elements.

Challenges of cell therapy include the harvesting of the appropriate cell populations and the expansion or isolation of sufficient cells for one or multiple patients. Cell harvesting may require specific media to maintain the stem cells ability to self-renew and mature into the appropriate cells. Ideally, “extra” cells are taken from the individual receiving therapy. Those additional cells can expand in culture and can be induced to become pluripotent stem cells (iPS), thus allowing them to assume a wide variety of cell types and avoiding immune rejection by the patient. The long-term benefit of stem cell administration requires that the cells be introduced into the correct target tissue and become established functioning cells within the tissue. Several approaches are being investigated to increase the number of stem cells that become established in the relevant tissue.

Another challenge is developing methods that allow manipulation of the stem cells outside the body while maintaining the ability of those cells to produce more cells that mature into the desired specialized cell type. They need to provide the correct number of specialized cells and maintain their normal control of growth and cell division. Otherwise, there is the risk that these new cells may grow into tumors.

In most fields, funding for basic or applied research for gene and cell therapy is available through the National Institutes of Health (NIH) and private foundations. These are usually sufficient to cover the preclinical studies that suggest a potential benefit from a particular gene and cell therapy. Moving into clinical trials remains a huge challenge as it requires additional funding for the manufacturing of clinical grade reagents, formal toxicology studies in animals, preparation of extensive regulatory documents, and costs of clinical trials. Biotechnology companies and the NIH are trying to meet the demand for this large expenditure, but many promising therapies are slowed down by a lack of funding for this critical next phase.

What are the ethical issues associated with gene and cell therapy?

Several ethical issues can arise during the development of any novel therapeutic. The development of genetic and cellular therapies share many ethical issues with other types of therapy, such as prosthetics, drugs, organ transplantation, and protein replacement. In addition, there are ethical issues unique to gene and cell therapy. In all cases, scientists, clinicians, regulatory committees, and concerned citizens take an active role in addressing these issues.

is important to any developing therapeutic. This is complicated by the fact that most gene therapy trials are Phase I trials, which means that safety of the vector and delivery mode are being evaluated and no direct benefit to the participant is expected. To assess potential benefit, regulatory committees often request that investigators administer a range of doses of the agent for the initial patients to determine whether higher doses do have adverse effects—even during Phase II/III trials. Thus, the dosage tested in a particular patient may be insufficient to induce a therapeutic response or may be so high as to cause toxicity.

raise the ethical question of whether these treatments will only be utilized by the wealthy. Biotechnology companies such as Novartis, who developed the leukemia treatment Kymriah, are aware of this and are developing programs to provide financial help to patients in the USA who are uninsured or underinsured. Another factor to consider is that gene and cell therapies are designed to be curative, and so the cost of therapy can be weighed against that of lifetime treatment. In the long-term, costs will likely be reduced by optimized production of cell and gene therapies and the development of therapies that do not need to be tailored to the individual. In the meantime, patient groups, clinicians, regulators and manufacturers all have a role to play in addressing the issue of cost.

with novel DNA sequences is a concern that may be considered in two ways. First, there is the issue of accidental contamination of the genome while conducting gene or cell therapy on somatic (adult) cells. To minimize the possibility of this, all vectors are tested to make sure they do not enter the germ line in experimental animals, and sperm from human males in clinical studies are tested to make sure the gene has not inserted in the genome. Second, there is the issue of intentional manipulation of the germline to alleviate disease. As new gene editing technologies have now made this much easier, there is currently much debate between scientists, clinicians, patient groups, and regulators regarding the ethics of editing, or not editing the human genome.

or human fetal tissue, as a source of stem cells remains an ethical issue. The development of stem cells from other sources such as iPSCs has somewhat reduced the dependence on ESCs.

What does in vivo and/or ex vivo gene therapy mean?

Clinicians and scientists may classify gene therapy according to whether the therapy is administered to cells in the body or out of the body. In vivo gene therapy means that therapy is administered directly the patient. The targeted cells remain in the body of the patient. With ex vivo gene/cell therapy the targeted cells are removed from the patient and gene therapy is administered to the cells in vitro before they are returned to the patient’s body.

What kinds of diseases do gene and cell therapy treat?

Characteristics of diseases amenable to gene therapy and cell therapy include those for which there is no effective treatment, those with a known cause (such as a defective gene), those that have failed to improve or have become resistant to conventional therapy, and/or cases where current therapy involves long term administration of an expensive therapeutic agent or an invasive procedure.

Gene therapy and cell therapy have the potential for high therapeutic gain for a broad range of diseases. An example would be those caused by a mutation in a single gene where an accessible tissue is available, such as bone marrow, and with the genetically modified cell ideally having a survival advantage.

Gene therapy and cell therapy also offer a promising alternative or adjunct treatment for symptoms of many acquired diseases, such as cancer, rheumatoid arthritis, diabetes, Parkinson’s disease, Alzheimer’s disease, etc.

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