Gene therapy.pdf

The full genome of the dodo has been sequenced, as well as the Nicobar pigeon, the dodo’s closest living relative. Cells that act as a precursor for ovaries or testes in the Nicobar pigeon have been shown to grow successfully in a chicken embryo. https://edition.cnn.com/dodo-de-extinction-mauritiu...

The full genome of the dodo has been sequenced, as well as the Nicobar pigeon, the dodo’s closest living relative. Cells that act as a precursor for ovaries or testes in the Nicobar pigeon have been shown to grow successfully in a chicken embryo. https://edition.cnn.com/dodo-de-extinction-mauritius-spc-intlscn/index.html They are now researching to see if these cells (called primordial germ cells, or PGCs) can turn into sperm and eggs. Gene therapy and cell therapy Gene Therapy and Cell Therapy Branch of regenerative medicine ➢process of replacing, engineering or regenerating human cells, tissues or organs to restore or establish normal function Gene therapy: delivery of therapeutic gene into a patient's cells to treat disease Cell therapy: delivery of intact, living cells into a patient to treat disease. Major types of gene therapy Gene replacement Corrects a cell with a non-functioning gene (loss-of-function variant) by introducing a functional gene Gene suppression Corrects a cell producing a toxic protein by targeting the abnormal gene to reduce/prevent protein expression Gene editing Corrects a DNA variant by cutting and then repairing DNA to reduce, introduce, or correct the variant Blau &Springer NEJM 333:1204, 2000 2 types of gene therapy: adding a normal gene to correct a specific gene disorder Ex vivo ❑cells are removed from the body ❑gene of interest is inserted into them ❑cells are cultured to increase cell numbers ❑cells returned to the body by infusion or transplantation (time consuming and expensive) In vivo ❑a gene is introduced directly into specific cells within the body (quick and inexpensive) ❑targeting certain cells (e.g., bone marrow stem cells) is difficult In vivo and ex vivo approaches to gene therapy Factors to be considered in gene therapy ❑How to deliver genes to specific cells, tissue and whole animals? (methods of delivery) ❑How much and how long the introduced gene will be expressed? ❑The site and dose of gene delivery ❑Are there any adverse immunological consequence of both delivery vehicle (Virus) and the gene in animals? ❑Are there any toxic effects? The ideal vector for gene transfer ❑High concentration of virus, allowing many cells to be infected or transduced ❑Convenience and reproducibility of production ❑Ability to transduce dividing and non-dividing cells ❑Ability to integrate into a site-specific location in the host chromosome, or to be successfully maintained as stable episome ❑A transcriptional unit that can respond to manipulation of its regulatory elements ❑Ability to target the desired type of cell ❑No components that elicit an immune response Gene transfer systems Gene therapy platforms Aside from being safe and cost-effective, an efficacious gene transfer system should be: ❑ able to target cell selectively ❑ transcriptionally competent for the desired length of time ❑ available in a highly concentrated active form ❑ immunologically neutral Why use viral vectors? ❑Virus are obligate intracellular parasites ❑Very efficient at transferring viral DNA into host cells ❑Specific target cells: depending on the viral attachment proteins (capsid or glycoproteins) ❑Gene replacement: non-essential genes of virus are deleted and exogenous genes are inserted Viral vectors in gene therapy ❑ Consider somatic vs germline gene therapy ➢ the later is currently banned! ❑ Gene therapy is limited to somatic cells and disorders that are caused by a single gene Figure 1. Trends in the rise and fall of AdV vectors in clinical trials of gene therapy and vaccine delivery for various diseases from 1993 to 2019. Adenoviral vectors ❑ds DNA viruses; usually cause benign respiratory disease ❑Can infect dividing, non-dividing cells ❑Replication-deficient vectors generated by replacing the E1 or E3 genes, which are essential for replication ❑Recombinant vectors are then replicated in cells that express the products of the E1 or E3 gene and can be generated in very high concentrations Clin & Trans Imm, Volume: 10, Issue: 10, First published: 12 October 2021, DOI: (10.1002/cti2.1345) Adenovirus as vectors Advantages • High titers • Both dividing and nondividing cells • Wide tissue tropism ➢ability of a pathogen to infect a specific organ or sets of organs • Can easily modify tissue tropism Limitations • Transient expression ( not good for genetic diseases) • Highly immunogenic ➢Immune response = reason behind short-term expression ➢elicits both cell-killing “cellular” response and antibody producing “humoral “ response ➢Humoral response results in generation of antibodies to adenoviral proteins • High titers of virus can be toxic • More suitable for cancer immunotherapy Adeno-associated viral vectors • simple, non-pathogenic, ssDNA virus dependent on helper virus (usually adenovirus) to replicate • 2 genes (cap and rep), sandwiched between inverted terminal repeats that define beginning, end of virus and contain the packaging sequence ❑cap gene encodes viral capsid proteins ❑rep gene product involved in viral replication, integration Viral DNA can integrate preferentially into human chromosome 19 • To produce an AAV vector, rep and cap genes are replaced with transgene • Total length of insert cannot exceed 4.7 kb, the length of WT genome Production of recombinant vector requires that rep and cap are provided in trans along with the helper virus gene products ❑ current method is to co-transfect 2 plasmids: one for vector, one for rep and cap into cells infected with adenovirus Adeno-associated viral vectors Advantages Disadvantages • Integration and persistent expression • No insertional mutagenesis • Can infect both dividing and nondividing cells • Safe • Size limitation: max 4.9 kb insert • Low titer of virus • Low level of gene expression Retroviruses (including Lentivirus, HIV and MMLV based vectors) • ssRNA genome • Lipid membrane enveloped • Host range determined by envelope protein • Random integration into host genome Retroviral vectors are based on Moloney murine leukemia virus (Mo-MLV), which can infect both mouse and human cells The retroviral genome Long Terminal Repeat (LTR): Necessary for integration into host genome ψ (Psi): packaging signal gag: Packages viral genome into viral particles pol: viral polymerase necessary for viral replication env: viral envelope proteins, necessary for entry into host cells, dictate host range Retrovitral vectors gag, pol and env are replaced with transgene of interest ❑expressed on plasmids in packaging cell line • Because the non-essential genes lack the packaging sequence, they are not included in the virion particle • To prevent recombination resulting in replication competent retroviruses, all regions of homology with the vector backbone is removed Lentiviruses Lentiviruses = subtype of retroviruses e.g., Human immunodeficiency virus (HIV), monkeys (SIV), and cats (FLV) Lentiviral-based vector systems were developed to overcome the deficits of early MLV-based retroviral systems Lentiviruses can infect both actively dividing and non-dividing cells, while retroviruses can only infect mitotic active cell types Design of replication incompetent lentiviral vectors (3rd generation) • Viral vector “gutted” as much as possible to create room for insert gene • 4-plasmid system: ❑Lentiviral vector ❑Two packaging plasmids (one with gag and pol, and a second with rev) ❑Also contains transfer and envelope plasmids Some 3rd gen systems have deletion w/in 3' LTR in the transfer plasmid, which is transcribed to the 5' LTR during reverse transcription ❑ results in a self-inactivating vector due to reduced promoter activity Lentiviral vectors The 3 plasmids containing viral genome components are transfected into the packaging line to create the infectious viral particles. Multiple plasmids are used so multiple recombination events would be required to reconstitute a replication competent virus. Risks associated with retroviruses: insertional mutagenesis Random integration of viral genome may disrupt endogenous host genes. Of special concern Is disruption of proto-oncogenes, which can lead to increased cancer risk Severe Combined ImmunoDeficiency (SCID) How is ADA deficiency treated? There are no real cures for ADA deficiency, but doctors have tried to restore ADA levels and improve immune system function with a variety of treatments: • Bone marrow transplantation from a biological match (for example, a sibling) to provide healthy immune cells • Transfusions of red blood cells (containing high levels of ADA) from a healthy donor • Enzyme replacement therapy, involving repeated injections of the ADA enzyme • Gene therapy - to insert synthetic DNA containing a normal ADA gene into immune cells 6-yr-old Ashanthi DeSilva-SCID sufferer treated with gene therapy-coloring at home in N Olmstead, OH (March 1993) Gene Therapy Gives Kids with SCID a Shot at a Normal Life Gene therapy in blood cells Bone marrow transplants replace the selfrenewing stem cells (the top cell) of a person with cancer or other disease with those of someone uninfected The goal is to have their body repopulate with healthy cells Therapeutic HIV protection gene Delivery strategies for in vivo gene therapy ”To the right tissue at the right dose at the right time” • Tissue specificity influenced by viral vector and promoter • Route of administration: IT, IV, ICM, local Delivery strategies for in vivo gene therapy Viral vs. non-viral delivery Gene therapy vectors Vector Advantages Disadvantages Retrovirus • High efficiency transduction of appropriate target cells • Long-term expressionintegration into chromosomal DNA • Potential for insertional mutagenesis • Requires dividing cells • Limited size of DNA insert Adenovirus • • • • • Transient expression • Immunogenicity • Direct cytopathic effects of virus • No infectious risk • Completely synthetic • No limitation on insert size • Potential for insertional mutagenesis if integration not site-specific • Limited size of DNA insert • Low efficiency • Limited target cell range • Transient expression Adenoassociated virus (AAV) Non-viral vectors eg, liposomes High transduction efficiency. Broad range of target cells Does not require cell division. Low risk of insertional mutagenesis • Does not require cell division • Site specific integration Approved gene therapies Vector Mechanism AAV vector Replace SMN1 Lentiviral vector Replace -globin AAV vector Replace RPE65t AAV1 vector Replace LPL Gammaretrovirus Replace ADA Lentiviral vector CD19 CART Engineered MSCV retrovirus CD19 CART Oncolytic virus Oncolytic virus Brexucabtagene autoleucel TECARTUS® Lisocabtagene maraleucel BREYANZI® Replication-incompetent retrovirus Mantle cell lymphoma/ALL Cancer 2020 CD19 CART Lentivirus Large B-cell lymphoma Cancer 2021 CD19 CART Idecabtagene vicleucel ABECMA® Lentivirus Relapsed/refractory myeloma Cancer 2021 BCMA CART Ciltacabtagene autoleucel CARVYKTI® Lentivirus Relapsed/refractory myeloma Cancer 2022 BCMA CART Papanikolaou and Bosio, Front Gen Editing, 2021 https://www.nytimes.com/20 23/10/31/health/sickle-cellfda-cure-crispr.html?s=09 Vertex and CRISPR Therapeutics focused on the gene-editing system CRISPR-Cas9. To treat sickle cell, CRISPR snips a piece of DNA in bone marrow stem cells. That frees a blocked gene to make a form of hemoglobin that normally is produced only by a fetus. The fetal gene directs the production of hemoglobin that does not form into the sickle shape. In clinical trials, patients no longer had the complications of sickle cell disease and no longer needed blood transfusions. Patients first have eight weeks of blood transfusions followed by a treatment to release bone marrow stem cells into their bloodstream. The stem cells are then removed and sent to the companies to be treated. Next, patients receive intense chemotherapy to clear their marrows for the treated cells. The treated cells are infused back into the patients, but they have to remain in the hospital for at least a month while the new cells grow and repopulate their marrows. Key concerns and related ethical concepts • • • • • Safety (nonmaleficence) Efficacy (beneficence) Informed consent (autonomy) Allocation of resources (justice and equity) Respect for human dignity “.. if we could make better human beings by knowing how to add genes, why shouldn’t we do it?” James Watson, 3/98 Arguments for germline gene therapy • Medical utility - the potential of a true “cure” • Medical necessity - may be only way to cure some diseases • Prophylactic efficacy - better to prevent a disease rather than to treat pathology • Parental autonomy - parents can make choices about what is best for their children • Easier, more effective, less risky than somatic gene therapy • Eradication of disease in future generations • Foster scientific knowledge • Part of being human - supporting human improvement “I’m absolutely for it [germline gene therapy] on the most fundamental of grounds. And that’s the grounds of human nature… Germline gene therapy will be done because of human nature. None of us wants to pass on to our children lethal genes if we can prevent it.” W. French Anderson, Director of Gene Therapy Laboratories, USC Arguments against germline gene therapy • • • • • • • Slippery slope - leads to misuse and abuse “eugenics” Lack of informed consent - fetus/embryo cannot consent Unknown/unforeseeable risks to individual, their offspring Violates genetic integrity of future generations Less “risky” alternatives exist Too costly - poor/misguided use of scarce resources Should not be attempted until more success in somatic gene therapy • Will widen the gap between the haves and have-nots • Devalues sense of “humanness” • Is playing god Other applications of recombinant vectors Vaccines! e.g., SARS-COV-2 List of special topics for group reporting • Synthetic biology • design and synthesis of molecular machines • Biobanking • Artificial intelligence approaches in genomics • Biomanufacturing • Regenerative medicine • Current genomics applications available in the Philippines • Other topics on new and emerging approaches in biochemistry and biotechnology Schedule: • January?