Gene Therapies - AHW PDF

GENE THERAPIES Biopharmaceuticals, KIMN10 Axel Hyrenius Wittsten Researcher Synthetic Immunology BMC C13 [email protected] Who am I? Cell-based Immunotherapy of solid tumors...

GENE THERAPIES Biopharmaceuticals, KIMN10 Axel Hyrenius Wittsten Researcher Synthetic Immunology BMC C13 [email protected] Who am I? Cell-based Immunotherapy of solid tumors Postdoc Functional and Molecular Cell-based Immunotherapy studies of leukemia of tumors UCSF USA PhD PI Lund University Lund University Sweden Sweden Lecture Objectives: MAIN Understand the overall concept and width of Gene Therapy Learn the basis of the main systems used for gene delivery/correction Be able to identify advantages/disadvantages/limitations for each system “Supplementary” Get insight into the clinical potential of Gene Therapy Start to grasp the capabilities and ethical concerns Get excited! Lecture Content: PART I General introduction and stratification of Gene Therapy PART II Detalied explanation of employed systems and their use PART III Example: Proof-of-principle to Clinical Trial Definition of Gene Therapy Gene Therapy is the insertion, alteration, or removal of genes within an individual's cells and tissues to treat disease *still controversy as to what should be included (definition in evolution) Suppression with RNAi or antisense oligonucleotides Increased/restored expression by introducing a gene Gene modification(s) with genome editing Classifying Gene Therapy Origin of Gene Therapy Proposed "that exogenous good" DNA could be used to replace the defective DNA in those who suffer from genetic defects. First gene therapy clinical study in 1990 at the NIH: - 4 year old girl (Ashanti DeSilva) - treatment for a genetic defect causing an immune system deficiency (ADA-SCID). Terminology Transformation = transfer of naked DNA into bacteria. Transfection = transfer of naked DNA into cells. Infection = infect with a wild type virus. Transduction = foreign DNA introduced into another cell via a viral vector Titer = way of expressing concentration of viral particles (Infectious Units per ml, IU/ml) Genetic background of diseases Environmental factors: infectious diseases Genetic factors: inherited disorders Monogenic (caused by one gene) Polygenic (requires many genes to manifest) ← uncommon Environmental factors + genetic factors: cancers, chronic diseases Types of faulty genetics Basis of Gene Therapy A ‘carrier vector’ is typically used to deliver the therapeutic gene to the patient's target cells. Majority (not all) of vectors are virus that have been genetically altered to carry normal human DNA. Gene delivery Virus have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner, we can take advantage of this capability and manipulate the virus genome to insert therapeutic genes. The vector is then inserted into cells and unloads its genetic material containing the therapeutic human gene into the target cell and the generation of a functional protein product from the therapeutic gene restores the target cell to a normal state. Ex vivo or In vivo lonzabioscience.com Ex Vivo: Self or non-self lonzabioscience.com Ex Vivo: Self or non-self AUTOLOGOUS ALLOGENEIC Larger-scale manufacturing possible Patient-specific Donor can be screened for desirable No rejection by the immune system characteristics Advantages No risk for graft-vs-host disease More reproducible manufacturing (less donor Repeated doses possible variability) Banking possible (immediate availability) High costs for manufacturing and Risk for graft-vs-host disease needs to be quality testing minimized through additional steps (cost increase) Challenges Starting material variability (donor Risk of rapid rejection as cells are still recognized variability) as foreign cells Delivery Vehicle: Non-viral Toualbi. Int J Mol Sci. 2021 Non-viral example Hereditary variant transthyretin amyloidosis (ATTRv) is a rare genetic condition where mutant TTR protein misfolds, forming aggregates which deposit as amyloid in various organs and tissues in the body. Patisiran is a liposomal siRNA specifically targeting TTR, reducing the accumulation of TTR in tissues, with subsequent improvement in clinical symptoms. Akinc. Nat Nanotechnol. 2019 Suhr. Orphanet J Rare Dis. 2015 Non-viral example P Y R A H E E T E N G AS E D S I FI A S C L O T N Szabó. Mol Ther. 2022 Plasmids: Excellent gene carriers Plasmid: Circular double stranded DNA molecule, can be propagated in bacteria Nucleoid Joshua Lederberg Nobel prize 1958 Bacterial Plasmids chromosome Can replicate independently of chromosomal DNA in the cell Naked DNA (i.e. no surrounding membrane) sciencelearn.org.nz/ Plasmid production Bacterial transformation is a process of horizontal gene transfer by which some bacteria take up foreign genetic material (naked DNA) from the environment. Plasmid mediated gene delivery Low gene transfer efficiency Only transient expression (non-integrating) Difficult with primary cells VIRAL VECTORS Delivery Vehicle: Viral biovian.com/news/viral-vector-and-gene-therapy-basics-summarized/ Viral vectors: Non-integrating virus Adenovirus: Class of virus with double-stranded DNA genomes that cause respiratory, intestinal, and eye infections in humans (e.g. common cold) Their DNA does not integrate into the host cell genome Adeno-associated virus: Class of small, single-stranded DNA virus that are not pathogenic to humans. Replication- defective. The DNA does not integrate into the genome of the host cell (it can integrate but at very low frequencies). Viral vectors: Integrating virus Retrovirus: Class of virus that can create double-stranded DNA copies of their RNA genomes. These copies can be integrated into the chromosomes of host cells. Most retrovirus can only infect dividing cells. Lentivirus (e.g. HIV): Sub-species of Retrovirus Unique ability among retroviruses of being able to infect non-dividing cells Adenoviral vectors 1) Class of virus with double-stranded DNA genomes that cause respiratory, intestinal, and eye infections in humans (e.g. common cold) 2) The transferred adenoviral genome does not integrate into the host genome, so gene expression is transient (episomal). Coughland. Front Immunol. 2020 Adenoviral vectors: Delivery Coughland. Front Immunol. 2020 Adenoviral vectors: Major positive features Pros 1) High efficiency of transduction in vitro and in vivo 2) High levels of transgene expression 3) Easily produced in high amounts (1011 IU/ml) 4) Infect both dividing and quiescent cells 5) Very rarely integrates into the host cell genome Adenoviral vectors: Major negative features Cons 1) Ad5 (most popular serotype) based vector infection requires expression of the coxackie adenovirus-receptor (CAR) on the cell surface for binding. However, most cell types do not express CAR. 2) Since most individuals have been exposed to Ad5, they will react with a neutralizing immune response to the virus, this is why in vivo gene transfer and repeated administration is not recommended. 3) Persistent gene expression is difficult to achieve in dividing cells due to absence of integration, and in non-dividing cells episomal DNA is degraded Adeno-associated vectors (AAV) 1) Class of small, single-stranded DNA virus that are not pathogenic to humans. 2) The DNA does not integrate into the genome of the host cell (it can integrate but at very low frequencies). AAV: Major features 1) Smaller than Adenovirus (1/3 of the diameter), have a relatively small packing capability 2) Can transduce both dividing and non-dividing cells 3) Low immunogenicity and cytotoxicity 4) Transduce cells as double stranded circular episomes (chromosomal integration can also occur) AAVs way to the Clinic Caveat: As many as 50% of patients are currently excluded from treatment due to pre-existing immunity to the viral capsids. Dunbar et al. Science. 2018 Clinical examples: AAV Different serotypes (AAV-1 through to AAV-11) Clinical trials: - Hemophilia B - Retinal disease - Parkinson’s disease - Cystic fibrosis - Lysomal storage diseases Issa. Cells. 2023 Clinical examples: Hemophilia B Hemophilia B is an inherited bleeding disorder resulting from a deficiency of clotting factor IX. When factor IX levels are insufficient, blood cannot clot effectively to stop bleedings. Requires frequent (at least once per week) intravenous protein replacement therapy of the deficient clotting factor. Lillicrap. Cell. 2017 Viral vectors: Integrating virus LTR: Long Terminal Repeat – Sequences that regulate viral transcription (i.e. promoter and poly-adenylation signal) Ѱ: Psi, Packaging signal gag: viral structural proteins pol: for replication and integration env: envelope proteins, mediate entry to cells. Recombinant integrating vectors Recombinant viral vector Separating viral components on “packaging” plasmids and the transgene on a “transfer” plasmid Producing integrating vectors Examples of integrating vectors Retroviral vectors: Major features 1) The vector genome integrates into the host cell genome, whereby persistent transgene expression is expected. 2) Successful transduction of host cells by gammaretroviral vectors requires cells to be dividing, therefore non-dividing cells “resistant”. 3) Due to the genome integration, there is a potential risk of Insertional mutagenesis. Insertional mutagenesis Adapted from Bushman. Mol Ther. 2020 Historical overview of HSC gene therapy Dunbar et al. Science. 2018 Clinical examples: Retrovirus Primary Immunodeficiences (PIDs) X linked SCID ADA SCID Wiskott Aldrich Syndrome Chronic Granulomatous disease Clinical example: ADA-SCID ADA-SCID - a rare disorder, approx 15 cases/year in Europe. Caused by a mutation that results in the absence of protein called adenosine deaminase (ADA), which is required for the production of lymphocytes. Children born with ADA-SCID do not develop a healthy immune system so they cannot fight off everyday infections, which results in severe and life-threatening illness. Without prompt treatment, the disorder often proves fatal within the child’s first year of life. ADA-SCID The main symptoms of ADA deficiency are pneumonia, chronic diarrhoea, and widespread skin rashes. Affected children also grow much more slowly than healthy children and some have developmental delay. Most individuals with ADA deficiency are diagnosed with SCID in the first 6 months of life. ADA-SCID: Gene therapy approach Treatment of ADA-SCID: Matched Related Bone Marrow Transplant (BMT) Or Matched Unrelated BMT Enzyme Replacement Therapy: Weekly injections with PEG- ADA Enzyme High et al. NEJM. 2019 Lentiviral vectors: Major features 1) The vector genome integrates into the host cell genome, whereby persistent transgene expression is expected. 2) Successful transduction of host cells by lentiviral vectors does not require active cell division because of nuclear transport mechanisms. 3) Due to the integration, there is still a potential risk of insertional mutagenesis, although less than with gammaretroviral vectors, because it is more random. Safety: Lentiviral vectors Lentivirus tends to integrate more within active genes, but less frequently upstream of transcriptionally active promoters. Lentiviral vectors may be less likely to activate adjacent oncogenes.  Update: Self-Inactivating (SIN) vectors Removal of endogenous strong enhancer elements using a “self-inactivating” design is another approach to decrease the risk of genotoxicity Applies to both lentiviral and gammaretroviral vectors Holst Wolff et al. J Biomed Sci. 2022 Safety concerns (real examples) 1) Gelsinger had partial ornithine transcarbamoylase (OTC) deficiency and was given an infusion of corrective OTC gene (adenoviral vector). He experienced a severe immune reaction to the vector and died 4 days after receiving the injection. Jesse Gelsinger 2) The two first gene therapy clinical trials (1st gen. retroviral) in patients with X-SCID showed exceptional results, most patients showing good long-term immune reconstitution and high overall survival of 90%. However, six patients unexpectedly developed T cell leukemia 2–14 years after the treatment (one fatal). 3) X-linked myotubular myopathy (XLMTM) is a severe congenital disease characterized by profound muscle weakness, with no currently approved therapies. As of October 2021, four children had died in a gene therapy trial (AAV) trial after experiencing liver failure linked to the treatment. Gene editing tools Adli. Nat Comm. 2018 CRISPR/Cas9 system In just a few short years, CRISPR/Cas9 has had a massive impact on scientific research, contributing to breakthroughs in medicine and biotechnology. CRISPR/Cas9 has the potential of introducing revolutionary changes in medicine, especially in treatment of genetic diseases Emmanuelle Charpentier & Jennifer Doudna Nobel prize 2020 CRISPR/Cas9 origins Biorender.com CRISPR/Cas9 way to the clinic Adapted from Fatih Rasul. Mol Cancer. 2022 CRISPR/Cas9 versions Adli. Nat Comm. 2018 CRISPR/Cas9 administration Van Hasteeren. Nat Biotechnol. 2020 CRISPR/Cas9 delivery methods (ex vivo) Sundaresan. FEBS J. 2023 CRISPR/Cas9 example Sickle cell disease (SCD) and thalassemia are among the most common inherited diseases, affecting millions of persons globally. They are caused by errors in the genes for hemoglobin, a substance responsible for carrying oxygen within the red blood cell. These mutations cause an imbalance between the chains of hemoglobin, which causes ineffective erythropoiesis. The resulting diseases are serious and, at times, fatal. Frangoul et al. NEJM. 2021  CRISPR/Cas9 example CRISPR Therapeutics Frangoul et al. NEJM. 2021 CRISPR/Cas9: what’s cooking? innovativegenomics.org Clinical examples: Acquired disease Cancer Introduction of tumor-specific T cell receptors Immune cells and Cancer The Chimeric Antigen Receptor (CAR) CARs differ from the T cell receptor Anguela and High. Annu Rev Med. 2019 What is CAR-T cell therapy? CAR-T cell CAR-T cell therapy involves re-engineering a patient’s own T cells to recognize and eradicate cancer. These T cells are genetically altered to express artificial receptors which enable the T cells to bind to a specific antigen on the patient’s tumor Tumor cells and kill them. cell How are CAR-T cells produced CAR-T cell therapy in B cell Malignancies Adapted from cancercenter.com Patient ”Proof-of-Principle” 2012 2023 Diagnos: acute lymphoblastic leukemia Two relapses Stopped responding to conventional therapy First child treated with CAR-T cells emilywhiteheadfoundation.org Clinical Success of CAR T cell therapy Example of CAR T-cell therapy approved by the FDA: KYMRIAH® Tisagenlecleucel. (2017) Approved for children and adolescents with relapsed or refractory acute lymphoblastic leukemia and adult Lymphoma. In the initial study (B-ALL) all signs of cancer disappeared in 27 of the 30 patients treated, and many had complete and long-lasting remissions. CAR-T cell therapy: Major features Pros Cons 1) When using patient’s own cells, no risk 1) High cost (personalized) of graft-versus-host disease 2) Length of time required for T-cell 2) Potential for lasting immunity even after processing and modification a single infusion 3) Adverse events including cytokine release syndrome (CRS) Broadening CAR-T cell Therapy Ivica et al. Healthcare. 2021 Functional Challenges in CAR T cell therapy Next generation CAR-T cells: Proof-of-principle to Clinical Trial Multi-antigen recognition of tumors New class of synthetic environmental sensor Developing new clinically relevant circuit CAR-T cells Mesothelioma Heavily associated with asbestos. Continued worldwide increase in incidence (continued use in developing countries coupled with a long incubation). Very aggressive disease with very limited treatment options. Highly immunosuppressive TME, which likely contributes to therapy resistance. Comes in three general subtypes: Epithelioid (mOS ~13 months), sarcomatoid (mOS ~4 months), and biphasic (mOS ~8 months). ALPPL2: a new tumor-specific antigen Combinatorial targeting using ALPPL2 Data from the Human Protein Atlas Combinatorial targeting using ALPPL2 SynNotch CAR-T cells exhibit superior efficacy in vivo ALPPL2 synNotch for other tumor types Next step toward the clinic:  Problems with SynNotch Adapted from Wagner et al. Nat Rev Clin Oncol. 2021 New humanized version Current status: ClinicalTrials.gov Identifier: NCT05617755 Concluding remarks: The rate of technological innovation of gene and cell therapy is significantly outpacing the ability to safely and expeditiously move promising candidates forward in order to benefit patients. Consequently, one of the greatest changes in the field of cell and gene therapy in the near future will be in the area of regulatory sciences and accommodating the unique challenges posed by these innovative technologies as we move forward toward personalized therapies. Cited from: Bulaklak et al. Nat Commun. 2020 Three essential tools for human gene therapy Dunbar et al. Science. 2018 Suggested Reading: Gene Therapy Overiews: Gene therapy comes of age (doi.org/10.1126/science.aan4672) The Promise and the Hope of Gene Therapy (doi.org/10.3389/fgeed.2021.618346) The Death of Jesse Gelsinger, 20 Years Later (tinyurl.com/yckkfzp) Gene Editing CRISPR technology: A decade of genome editing is only the beginning (doi.org/10.1126/science.add8643) CAR-T cells: Chimeric Antigen Receptor Therapy (doi.org/10.1056/NEJMra1706169) The Principles of Engineering Immune Cells to Treat Cancer (doi.org/10.1016/j.cell.2017.01.016)