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Gene and Cell therapy (Vectors for gene delivery (in vivo) (Herpes simplex…
Gene and Cell therapy
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CRISPR
CRISPR-Cas9
A small piece of RNA with a short "guide" sequence is made to recognise and bind to a specific target sequence of DNA. The RNA also binds to the Cas9 enzyme, which cuts the DNA at the targeted location. The cell's DNA repair machinery is then used to add or delete pieces of genetic material, or to make changes to the DNA by replacing an existing segment with a customised DNA sequence. (Link)
Cas9 is the enzyme that is used most often (Link)
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CRISPR-Cas3
Cas3 breaks down bacterial DNA. This action turns the CRISPR-based bacterial immune system on itself, prompting the cell’s death (Link)
CRISPR-Cas10
CRISPR-Cas10 is superior at targeting rapidly mutating viruses. While CRISPR-Cas9 requires specific DNA sequences on the virus to be intact in order to cut it, the researchers found that CRISPR-Cas10, in the bacteria Staphylococcus epidermidis, can mount an effective immune response even when mutations are present in these sequences (Link)
CRISPR-Cas10 lacks a PAM sequence and instead looks more broadly for sequences that more or less match it. This allows it to instigate a strong and immediate immune response to invading viruses without having to have a number of guides to be made (Link)
CRISPR-Cpf1
CRISPR-Cpf1 is smaller than Cas9, it has different sequence requirements and is highly specific. Cpf1 exhibits a lower rate of cleavage for the Cpf1 PAM sequence, but off-target mutagenesis rarely happens (Link)
CRISPR-C2c2
Targets RNA rather than DNA — a feature that holds potential for studying RNA and combating viruses with RNA genomes (Link)
Epigenetics
Any process that alters gene activity without changing the DNA sequence, and leads to modifications that can be transmitted to daughter cells. Includes methylation, chromatin modification, acetylation, phosphorylation, ubiquitylation, and sumolyation (Link)
Epigenetic change is a regular and natural occurrence but can also be influenced by several factors including age, the environment/lifestyle, and disease state. It can result in diseases like cancer. At least three systems including DNA methylation, histone modification and non-coding RNA (ncRNA)-associated gene silencing are currently considered to initiate and sustain epigenetic change (Link)
Methylation inhibiting drugs, bromodomain and inhibitors, HAT inhibitors, protein methyltransferase inhibitors, histone methylation inhibitors, and HDAC inhibitors are all examples of epigenetic drugs (Link)
Azacytidine has been approved for MDS (myelodysplastic syndrome) in 2004 and Decitabine for MDS in 2006, both are DNA methyltransferases (Link)
Ruxolitinib, a Janus Kinase 1 and 2 inhibitor, was approved by the FDA for Myelofibrosis in 2011 (Link)
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DNA hypomethylation can activate oncogenes and initiate chromosome instability. DNA hypermethylation initiates silencing of tumor suppressor genes. (Link)Global histone modification patterns are also found to correlate with cancers such as prostate, breast, and pancreatic cancer. Subsequently, epigenetic changes can be used as biomarkers for the molecular diagnosis of early cancer (Link)
Methyl donors and histone deacetylase (HDAC) inhibitors have been investigated for possible therapeutic effects to rescue memory and cognitive decline found in neurodegenerative diseases (Link)
US FDA approved HDAC inhibitors Vorinostat (suberoylanilide hydroxamic acid) in 2006 and Istodax (romidepsin) in 2009 for the treatment of hematological malignancies (Link)
Cytidine analogs 5-azacytidine, zebularine, and nucleoside analogs are inhibitors of DNA methylation which can sequester DNA methyltransferase enzymes after being incorporated into DNA. These have been well characterized and are approved for use in pre-clinical and clinical trials for the treatment of cancer (Link)
RNAi - Gene Silencing
- Trigger RNA (dsRNA or miRNA primary transcript) is processed into an short interfering RNA (siRNA) by the RNase II enzymes Dicer and Drosha
- siRNAs combine with Argonaute (Ago) and other proteins to form an RNA-induced silencing complex (RISC)
- The siRNA is unwound during RISC assembly and the single-stranded RNA binds to the complementary mRNA target
- This causes degradation of the target transcript or inhibition of translation
(Link)
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Onpattro (Patisiran) became the first RNA interference drug approved by the EMA and FDA in August 2018. Onpattro is used to treat hereditary transthyretin amyloidosis, a rare disease that causes accumulation of misshapen amyloid proteins. This damage tissues and organs and can lead to organ failure and death. (Link)
Germ line Gene Therapy
Healthy gene is introduced into a reproductive cell e.g. sperm or egg, making the change heritable (Link)
These therapies are very controversial and there are no cases of germ line therapies undergoing clinical trials (Link)
Stem cell therapy
Most extensively used stem cell treatment is hematopoietic (or blood) stem cell transplantation, e.g. bone marrow transplantation, to treat certain blood and immune system disorders or to rebuild the blood system after treatments for some kinds of cancer (Link)
Some bone, skin and corneal (eye) injuries and diseases can be treated by grafting or implanting tissues, and the healing process relies on stem cells within this implanted tissue (Link)
All stem cells can self-renew and differentiate into more specialized cells. (Link)
There are many different types of stem cells that come from different places in the body or are formed at different times in our lives.
- Embryonic stem cells that exist only at the earliest stages of development
- Various types of tissue-specific
- (Adult) stem cells that appear during fetal development and remain in our bodies throughout life
(Link)
Induced pluripotent stem (iPS) cells are cells that have been engineered in the lab by converting tissue-specific cells, such as skin cells, into cells that behave like embryonic stem cells. (Link)
A whole-body graft of genetically modified stem cells was used in November 2017 to treat a severe form of epidermolysis bullosa (EB), an often-fatal group of conditions that cause skin to blister and tear off at the slightest touch (Link)
They extracted and cultured the patients own naturally occurring stem cells. Then infected those cells with a retrovirus bearing healthy copies of the needed gene, and grew them into sheets ranging from 50 to 150 square centimetres (Link)
Performed by/ lead investigator Michele De Luca, M.D., a regenerative medicine specialist at the University of Modena and Reggio Emilia, Modena, Italy (Link)
Holoclar, the first stem cell therapy approved in the EU, only around 1,000 people annually in the whole of Europe will be eligible: burn victims who have become blind but whose eyes have not been too extensively destroyed (Link)
Ex vivo Gene Therapy
Genetic material is extracted from a patients bone marrow/ target tissue and manipulated in vitro. The altered genetic material is then transducted and/or transfected into the target tissue. (Link)
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Zinc Finger Nucleases
Zinc finger DNA-binding proteins (ZFPs) recognize and bind to specific sequences of DNA. They can be engineered to bind anywhere. Nucleases, a functional domain that cuts DNA, are attached to ZFPs, to make a zinc finger nuclease (ZFN) (Link)
ZFNs can knock out a gene or insert a therapeutic gene into the genome in a targeted fashion. (Link)
If cells are only treated with ZFNs, the repair process frequently results in the rejoining of the two broken ends of the DNA, which disrupts the original DNA sequence and therefore also gene function. (Link)
A therapeutic gene can be inserted if cells are treated with ZFNs in the presence of an additional “donor” DNA sequence. (Link)
Sangamo and Pfizer developed zinc finger protein transcription factors (ZFP-TFs) treat ALS, which was the first gene editing therapy to be used in human in November 2017 (Link)
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Non-viral gene delivery
Clinical trials to inject naked DNA plasmids or naked PCR products have been performed. This method is safe and simple, but low gene delivery efficacy (Link)
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Sonoporation: ultrasonic frequencies to disrupt cell membrane (Link)
Magnetofection; magnetic fields are used to concentrate particles containing nucleic acid into the target cells (Link)
Gene guns (shoots DNA coated gold particles into
cells by using high pressure) (Link)
Receptor mediated gene transfer: DNA is conjugated with specific proteins or liposomes or both. Liposomes undergo endocytosis to give transient gene expression (Link)
Lipoplexes (made up of anionic and neutral lipids) and polyplexes (complex of polymers with DNA) have been used to facilitate delivery of the DNA into cell (Link)
CAR Therapy
Engineered T cells express a Chimeric Antigen Receptor (CAR) on their cell membrane. The CARs connect with an external target-binding domain designed to recognize a specific tumor antigen and an internal activation domain responsible for activating the T cell when the CAR-T bind its target (Link)
Second and third generation CARs have additional costimulatory domains that further enhance the immune response (Link)
Often used in cancer treatments- T cells are engineered to enhance the response from the immune system against a specific tumor antigen (Link)
CAR therapy starts with the extraction of T cells from the own patient, a process called leukapheresis. The T cells are then genetically modified to express a CAR and expanded in vitro. Finally, they are reinfused into the patient, ready to fight the tumor (Link)
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Patients receiving CAR therapy can experience strong cytokine release syndrome (CRS), Juno Therapeutics had to terminate its lead CAR program (March 2017) after a total of 5 patients died of cerebral edema (Link)
Companies with ongoing CAR therapy clinical trials include Juno/ Celgene and Mustang Bio in the US, Celyad in Belgium and the French Servier in partnership with Pfizer (Link)
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