Understanding Gene Therapy

What is Gene Therapy?

• Gene therapy focuses on protein-based therapies, wherein the patient lacks a particular protein or has a mutated version of it, like in sickle cell anemia
• Treatment involves replacing the missing or mutant protein
• Traditional treatments require constant dosing, but gene therapy seeks to permanently or semi-permanently reverse the deficiency
• Gene therapy now includes prevention therapies like vaccines
• Gene therapy introduces genetic material (DNA) into a patient's cells
• The genetic material can be a healthy copy of a therapeutic gene, a means to fix a faulty gene, or a method to switch off or modulate the expression of a 'bad' gene
• Ideally, gene therapy is targeted to specific cell types, but that remains challenging
• It may only require one dose to treat a patient, depending on the disease and method of delivery
• Delivery is the biggest challenge, requiring the genetic material to reach the right cells while evading the immune system
• Viruses are the most commonly used vectors for delivery, although this is changing
• Viruses are utilized for the delivery of genetic material to host cells, but the viruses used in gene therapy are modified

Types of Gene Therapy

• In vivo gene therapy delivers genetic material directly into the patient, where it is taken up by cells, using viral or non-viral methods for RNA or DNA delivery
• Ex vivo gene therapy (aka modified cell therapy) removes cells from the patient, introduces genetic material, and returns the modified cells, using viral or non-viral methods for RNA or DNA delivery
• A key challenge in both methods is effective delivery

Gene Therapy Pipeline

• The number of therapies in preclinical stage is around 1,500 in Q2 2023
• The number of therapies in phase I is around 240 in Q2 2023
• The number of therapies in phase II is around 260 in Q2 2023
• The number of therapies in phase III is 30 in Q2 2023
• The number of therapies in pre-registration around 6 in Q2 2023
• Total number of gene therapies stands at approximately 2,075 as of Q2 2023
• Oncology accounts for 54% of therapies, while non-oncology accounts for 46%
• In vivo genetic modification accounts for the majority of the therapies

Viral Properties for Gene Therapy

• Successful gene therapy requires the genetic material to enter the cells (and the nucleus if it's DNA)
• A delivery method is necessary
• Electroporation, the delivery of DNA to localized tissues, is not always suitable and not very efficient in vivo
• Chemical methods involves liposomes which can fuse with cell membranes
• Lipid Nanoparticles (LNPs) are also chemical methods

Viruses as Vectors for Gene Therapy

• Viruses are tiny, non-cellular parasites that infect virtually every type of known cell.
• They cannot perform metabolism without parasitizing a cell, meaning they are not considered alive outside of a host
• Different viruses are specialized for infecting particular species and cell types
• Viruses can replicate independently of a cell's chromosomes but not independently of the cells themselves
• They take control of the cell's metabolic machinery to replicate and they must invade a host cell to replicate
• Viruses can be classified as enveloped or non-enveloped

Approved Gene Therapies

• Luxturna is approved for inherited retinal dystrophy and involves adeno-associated virus
• Spinraza is approved for spinal muscular atrophy and involves adeno-associated virus
• Zolgensma is approved for spinal muscular atrophy and involves adeno-associated virus
• Imlygic is approved for melanoma and involves Herpes Simplex

Viral Vectors

• The main viral vectors in gene therapy includes retrovirus, lentivirus, Herpes Simplex Virus type 1, Adeno-Associated Virus, and Adenovirus
• Retrovirus and lentivirus integrate into the host genome
• Adeno-Associated Virus and Adenovirus are generally non-integrating, persisting predominantly as extrachromosomal episomes in the cell nucleus

How Viruses Enter a Cell

• HIV particles enter cells only if the virions bind to a membrane protein called CD4, found on T cells
• When the proteins in a virion's envelope bind to CD4, the lipid bilayers of the particle's envelope and the plasma membrane of the T cell fuse.
• The viral capsid can then enter the cytoplasm
• Viral genes required to proceed after entry will be stripped out of those used as Gene Therapy vectors

Viral Delivery Safety

• Gene therapy must ensure the vector virus will not cause disease
• The vector needs to have desirable delivery traits, and the undesirable replication traits should be removed
• Natural viruses are genetically modified to prevent replication in cells by removing critical genes
• The missing genes are added separately to the cells used to make the virus, along with the therapeutic gene

Adeno-Associated Virus

• AAV exhibits low immunogenicity and is non-pathogenic, works in both dividing and non-dividing cells
• It gives high gene expression level for a long time
• It has selective infection of many types of cells and does not typically integrate into the genome
• AAV has limited capacity for gene insertion, requires a high amount for efficient transduction
• Generating stable cell lines is technically demanding and it requires a helper virus or plasmid for replication
• AAV is made up of VP1,2 and 3 capsid proteins
• Each capsid contains 60 proteins
• Small amino acid variations dictate the type of cell/tissue that the virus infects (tropism), resulting in numerous serotypes

Production of viral gene therapies

• Transfection of plasmids containing genes necessary to 'build' a virus containing a therapeutic gene will form the vector
• Most common cell line used is HEK293
• This approach is the most common for AAV, Ad and lenti/retroviruses
• There is also a system for AAV production using insect cells
• Upstream requires plasmids, transfection on large scale, and a production phase of 3-5 days
• Some processes still rely on cell hosts that grow in attached culture
• AAV production involves optimizing harvest, transfection, virus recovery, extraction clarification, concentration
• The end product requires drug production in the form of filtration and processes involving purification on affinity, charge, and size

Challenges in AAV production systems

• Current production systems result in very high ratio of empty to full viral particles/capsids
• There is then a further sub-set that are not actually infectious when delivered
• High numbers of virus are required for effective dosing, typically, 10^13 vg per eye, or 5 x 10^13 vg/kg
• This raises concerns that high doses could elicit an immune response
• Technology is still quite underdeveloped

Non-Viral Gene Therapy

• Refers to the means of delivery, not the therapy itself
• DNA needs to reach the nucleus
• RNA does not need to reach the cell nucleus
• Ribonucleoprotein such as CRISPR-Cas9 needs to reach the nucleus

Plasmid DNA

• Plasmid DNA is injected into patient tissue, and can be naked or encapsulated in lipid-based particles
• It is less immunogenic than viruses
• An example is VM202 – pDNA encoding Hepatocyte Growth Factor (HGF)
• It treats Diabetic Peripheral Neuropathy using multiple injections into the calf muscle, and is shown to be safe with long lasting relief

RNA Therapeutics

• There are several types of RNA therapies, ASO, aptamer, RNAi and mRNA, which can Promote Degradation / Inhibit Translation; and Protein / Antigen Expression

mRNA Vaccines

• Conventional mRNA vaccines need to be delivered inside a Lipid coat
• Self Amplifying mRNA Vaccines delivers replicase genes

In Vitro Transcription

• In Vitro Translation requires no cells for production

Operation Warp Speed

• Pfizer/BioNTech and Moderna received $4.5bn from the U.S. government to accelerate SARS-CoV2 vaccine development
• Moderna generated the mRNA sequence for the spike protein after 2 days and the Phase I trial started after 67 days
• The FDA approved the vaccine after 11 months which included 8 months of clinical trials
• mRNA vaccines takes advantage of years of research into RNA therapeutics
• the detection and elimination of the mRNA by the immune system prevents pseudouridine (a modified version of UTP) detection and elimination

Delivery - Nanoparticles

• Naked RNA is unstable and is quickly degraded
• Encapsulating or complexing RNA with various types of polymers protects it by promoting entry into cells and escape from the endosome
• Lipid nanoparticles: encapsulate RNA
• Polymers: e.g. PEI. Taken up by endocytosis
• Nanoemulsions: Lipid shell around 'oily' core onto which RNA adsorbs