Gene therapy returns to centre stage Lab of molecular genetics Jehyun Nam

What is gene therapy? Gene augmentation therapy Gene inhibition therapy Killing of specific cells http://www.yourgenome.org/facts/what-is-gene-therapy

How is DNA transfer done? A section of DNA/gene containing instructions for making a useful protein is packaged within a vector. The vector acts as a vehicle to carry the new DNA into the cells. Once inside the cells of the patient, the DNA/gene is expressed by the cell’s normal machinery leading to production of the therapeutic protein and treatment of the patient’s disease. http://www.yourgenome.org/facts/what-is-gene-therapy

How is DNA transfer done?

Gene augmentation therapy This is used to treat disease caused by a mutation that stops a gene from producing a functioning product This therapy adds DNA containing a functional version of the lost gene back into the cell http://www.yourgenome.org/facts/what-is-gene-therapy

2. Gene inhibition therapy Suitable for the treatment of infectious disease, cancer and inherited disease caused by inappropriate gene activity The basis of this therapy is to eliminate the activity of a gene that encourages the growth of disease-related cells. http://www.yourgenome.org/facts/what-is-gene-therapy

3. Killing of specific cells Suitable for diseases such as cancer that can be treated by destroying certain groups of cells. The aim is to insert DNA into a diseased cell that causes that cell to die http://www.yourgenome.org/facts/what-is-gene-therapy

1. Haematopoietic-stem-cell gene therapy

1. Haematopoietic-stem-cell gene therapy Haematopoietic stem/progenitor cells (HSPCs) are isolated from the bone marrow (or mobilized peripheral blood) of a patient affected by a primary immunodeficiency — an inherited deficiency of a lymphocyte development gene that prevents formation of the lymphoid lineages. Following culture ex vivo in conditions that stimulate cell proliferation, the cells are exposed to a retroviral vector expressing a functional cDNA copy of the defective gene and then infused back into the patient after a few days. Infusion usually takes place following administration of a pharmacological conditioning regimen that eliminates the endogenous bone marrow progenitors and favours engraftment of the transplanted cells. The engrafted gene-corrected stem or progenitor cells generate functional progeny that reconstitute all lymphoid lineages and restore immune functions to the patient. If the gene-corrected cells have a selective growth advantage compared to the unmodified cells, full reconstitution of the immune cell compartments is obtained even from a few engrafted transduced progenitor cells, as depicted in the figure, and this may occur even without conditioning. If the engrafted progenitor cells have self-renewal capacity, they ensure long-term correction of the disease. If the engrafted cells are multipotent stem cells, they generate gene-marked cells in all haematopoietic lineages. NK cells, natural killer cells.

1. Haematopoietic-stem-cell gene therapy Gene therapy approaches for SCID. (A) Previously (4, 5), after informed parental consent, bone marrow was obtained from young boys with SCID-X1 who did not have an HLA-identical donor. Selection for the CD34+ cell population was performed, and these cells were stimulated to grow in media containing 4% fetal calf serum and supplemented with SCF, IL-3, Flt-3 ligand (FLT-3L), and megakaryocyte growth and differentiation factor (MGDF). The cells were transduced with a Moloney leukemia virus_based (MLV-based) replication-incompetent vector containing the common γ chain (γc) for 3 days. The cells were reinfused into the patients without a preparative conditioning regimen. In the majority of the boys, mature and functional T cells were reconstituted. Unfortunately, 3 of 11 boys enrolled in this clinical trial developed a T cell leukemia related to the oncoretroviral vector used (6). J Clin Invest. 2005;115(8):2064-2067. doi:10.1172/JCI26041. J Clin Invest. 2005;115(8):2064-2067. doi:10.1172/JCI26041.

2. Liver-directed gene therapy

2. Liver-directed gene therapy Nonintegrating Viruses Adeno-associated virus (AAV), a small virus that doesn’t cause disease and elicits only very minor immune responses. AAVs deliver their payloads without integrating them into the genome. Acts as a minichromosome.

3. T-cell immunotherapy for cancer T cells can be genetically engineered to recognize tumour-associated antigens in various ways in current clinical trials. If a patient expresses a tumour-associated antigen that is recognized by an available receptor structure, autologous T cells can be genetically engineered to express the desired receptor. New receptors can be generated in a variety of ways. a | T cells can be identified and cloned from patients with particularly good antitumour responses. Their T cell receptors (TCRs) can be cloned and inserted into retroviruses or lentiviruses, which are then used to infect autologous T cells from the patient to be treated.

3. T-cell immunotherapy for cancer T cells can be genetically engineered to recognize tumour-associated antigens in various ways in current clinical trials. If a patient expresses a tumour-associated antigen that is recognized by an available receptor structure, autologous T cells can be genetically engineered to express the desired receptor. New receptors can be generated in a variety of ways. a | T cells can be identified and cloned from patients with particularly good antitumour responses. Their T cell receptors (TCRs) can be cloned and inserted into retroviruses or lentiviruses, which are then used to infect autologous T cells from the patient to be treated. Chimeric antigen receptors (CARs) can be generated in a variety of ways. Most commonly, sequences encoding the variable regions of antibodies are engineered to encode a single chain, which is then genetically engrafted onto the TCR intracellular domains that are capable of activating T cells. These CARs have antibody-like specificities, which enable them to recognize MHC-nonrestricted structures on the surfaces of target cells.

Reasons for success in recent clinical trials Advances in vector manufacturing and characterization could be facilitating the increased transduction of target cells with lower adverse effects. In recent letiviral-vector-based HSC gene therapy In adoptive T-cell therapy: A subpopulation of T memory stem cells plays an important part in supporting long-term efficacy In liver-directed gene therapy: Detailed epitope-specific immune monitoring of antiviral T-cell responses on in vivo AAV gene delivery

Challenges ahead and prospective developments -targeting gene editing

Challenges ahead and prospective developments -Retinal gene therapy he eye, in particular, is an attractive target for gene therapy for several reasons. The eye is one of the few immunologically privileged sites in the body, so the gene vectors used are unlikely to cause a systemic immune response. Given the defined volume of the eye, small amounts of viral vectors may be all that are necessary to achieve therapeutic effects—likely to be a positive for reducing the risk of toxicity and increasing the likelihood of being able to manufacture quantities of vector sufficient to treat the retina. The eye also allows for localized treatment without intravenous delivery, thus decreasing the chance of systemic absorption and toxicity. Finally, the effects of localized ocular treatments can be easily observed and monitored for efficacy and safety, something that cannot be readily done with systemic conditions. With these advantages in using the eye as a target for gene therapy, and the continued understanding of gene mutations and their role in retinal diseases, investigators are actively determining the potential for gene therapy in different conditions (See Table 1). 

Challenges ahead and prospective developments -Retinal gene therapy he eye, in particular, is an attractive target for gene therapy for several reasons. The eye is one of the few immunologically privileged sites in the body, so the gene vectors used are unlikely to cause a systemic immune response. Given the defined volume of the eye, small amounts of viral vectors may be all that are necessary to achieve therapeutic effects—likely to be a positive for reducing the risk of toxicity and increasing the likelihood of being able to manufacture quantities of vector sufficient to treat the retina. The eye also allows for localized treatment without intravenous delivery, thus decreasing the chance of systemic absorption and toxicity. Finally, the effects of localized ocular treatments can be easily observed and monitored for efficacy and safety, something that cannot be readily done with systemic conditions. With these advantages in using the eye as a target for gene therapy, and the continued understanding of gene mutations and their role in retinal diseases, investigators are actively determining the potential for gene therapy in different conditions (See Table 1). 

Challenges ahead and prospective developments -Other relevant developments Delivery of transgenes to the brain by AAV or lentiviral vectors. Oncolytic viruses Adenoviral vectors of simian origin are also being assessed for their ability to induce humoral and cellular immunity through vaccination.

Future outlook Gene therapy enables the targeted delivery of information-rich gene-based cassettes that facilitate the stable, sustained and regulated expression of biological agents. Gene therapy directs powerful biological processes towards the goals of disease correction, tissue repair and regeneration. Improvements in vector manufacturing and characterization will allow the standardization and comparative assessment of vector performance between trials.