Gene Therapy

Diseases being targeted by gene therapy

• cancer

• monogenic disease

• infectious diseases

• cardiovascular diseases

Gene therapy strategies

• aim to deliver a gene in order to overcome disease/infection

  • non functional gene is replaced with functioning gene

• suicide gene

  • for cancer

  • use enzyme not found in cells, substrate binding = production of toxic substance that kills cells

• for a nonfunctional gene, deliver a gene which produces a product that blocks the expression of the faulty gene

• gene editing to correct defects

Gene therapy: In Situ Therapeutic Protein Production

• in situ - on site

• therapeutic proteins are hard to produce and expensive

• e.g. deliver anti-her2 mAb gene for herceptin for her-2 +ve cancer and get pt own cells to produce herceptin and release into systemic circulation

Suicide genes

• gene-directed enzyme pro-drug therapy (GDEPT)

• often uses a combination of HSV thymidine kinase and ganciclovir

• HSV TK has > 1000 fold greater efficiency in phosphorylating ganciclovir than mammalian kianses

  • not well phosphorylated normally

• incorporated into DNA as its similar to guanosine and induces apoptosis

nucleic acid based therapies

• decoy oligonucleotides - interfere with transcription of a gene by binding tot he promoter gene and stop it being expressed

• antisense oligonucleotide - stop translation by being complementary to a specific sequence and stopping the translation into protein that causes disease

RNA interference

• siRNA

• knock down gene expression if sequence is known

• binds to mRNA and induces cleavage which degrades mRNA

• reduce expression of gene at protein level

miRNA

• micro RNA

• derived from genes that specifically code mRNAs and not proteins

• important in regulating gene expression

• cause destabilisation and reduction of translation into protein instead of mRNA destruction

• over and underexpressed in many cancers

role of miRNA-34a

• involved in pathways that drive proliferation survival of cells

• regulates pathways that control cell proliferation, migration and invasion, resistance to apoptosis and immune evasion

• involved in cancers

• targets include androgen receptors (prostate), PDL-1, C-MYC and CD44

miRNA in clinic

• chemically modified to make stable in serum

• specific targeting

• inhibition of in vivo tumour growth

• caused complete cure in one animal

direct delivery of genes/nucleic acids into cells

• put therapeutic gene/oligonucleotide/miRNA in a suitable vector (virus)

• inject into patient

• targeting will allow it to reach the cell and delivery

cell-based delivery of genes/nucleic acids into cells

• take a patients own cells

• modify in vitro

• select for cells that have been modified and put them back in

could be used in sickle cell anaemia

challenges to deliver

• complex formation

• unstable in serum

• don’t get into cells well

• need to get through nuclear membrane to get into nucleus

  • may need to wait until dividing

• transport from delivery site

Retrovirus

• ssRNA genome

• 100nm in diameter

• some infect only dividing cells

  • lentiviruses can do it all

• can be used to target specific cells

• risk of immune response and tumour development

Lentiviruses

• randomly insert into genome

• if they insert into a tumour suppressor gene then they can stop it acting

• if they insert near a proto-oncogene they can turn it on

Adenovirus

• Infects dividing and non-dividing cells

• Doesn’t integrate

• Can be used to target specific cell types

• Possible immune response

Gendicine

• recombinant andenovirus with p53

• used to treat head and neck cancer

• used in combo with chemotherapy

Ocorine

• oncolytic virus - a virus that selectively infects and kills tumour cells

• Oncolytic viruses are engineered to take advantage of key regulatory factors of the cell cycle and the virus life cycle

• Oncorine can’t replicate in normal cells due to induction of p53, but can do so in p53-deficient tumour cells

• modified virus will proliferate in p53 deficient cancer cells and kill them

Adeno-associated viruses

• ssDNA genome

• Genome ~5 kbp

• ~20 nm diameter

• Infects dividing and non-dividing cells

• Specific integration (chr. 19)

• Can be used to target specific cell types

• Little immune response

Glybera

Adeno-associated virus serotype used as vector

• For the treatment of lipoprotein lipase deficiency (LPLD)

• Rare disease, affecting 1-2 people per million

• Causes dramatically increased fat levels in the blood

• Glybera was administered as a one-time series of small i.m. injections into the leg

Physical non-viral nucleic acid delivery methods

Physical

• direct

• gene gun

• ultrasound

• electroporation

Chemical non-viral nucleic acid delivery methods

for in vivo

• encapsulation with polymer

• complexation

Advantages and disadvantages of non-viral vectors

ADV:

• Easy to prepare and modify (e.g. targeted) because they’re synthetic

• Reduced immunogenicity

• Large capacity

DADV:

• Transient

• Poor efficiency

Types of non-viral vectors

• liposomes

• dendrimers

• cationic polymers

• cell penetrating peptides

Lipoplexes

DNA/RNA + liposome

interact with cell surface and get taken up into cell

Dendrimers

• Wide range of chemical approaches and functional groups

• Surface groups can be functionalized for targeting, stealth, etc

• Can be designed to release under specific conditions

• Amine-rich dendrimers for complexation with negatively charged nucleic acids (N means it’s positively charged)

Cationic Polymers

• mix with nucelic acid

• form a circular complex called toroid

Cell-penetrating peptides

• HIV TAT protein and Drosophila Antennapedia transcription factor shown to translocate cell membranes

• Many CPPs discovered to date - potential for delivery of a variety of cargoes (including liposomes, nanoparticles, etc for biological entities and traditional drugs)

• Low cytotoxicity, dose-dependent, can be conjugated to carriers

• Tend to be arginine rich (e.g. TAT (48-60) = GRKKRRQRRRPPQ)

• Various uptake mechanisms, including direct translocation and the different types of endocytosis

• Particularly promising for oligonucleotides - packaging and delivery in one

Potential for Non-viral vectors: what characteristics do we want ideally?

• Small

• Stable in serum

• Low toxicity

• Specific cell targeting

• Cytosolic release of cargo

• Nuclear targeting (depending on cargo)

• High transfection efficiency

Anti-angiogenesis strategies

• Anti-sense RNA or siRNA to block translation of GF or its receptor

• Oligonucleotides to block transcription of GF or its receptor

• Gene therapy to induce production of soluble receptor or mAb (against receptor of GF)

  • mop up existing GF and stops it binding to receptors on cells that drive angiogenesis

Glioblastoma tissue

• VEGF is highly up-regulated in GBM tissue and levels correlate to disease progression

• Gene for soluble VEGF receptor delivered in murine glioma model - mops up VEGF and reduces angiogenesis

RGD

• tripeptide

• targets intergrin receptors

• integrin receptors upregulated in cancers

• RGD binds to them in cancer cells and aids in targeting them to tumour cells

Haematopoietic Stem Cell Transplantation (HSCT)

For peripheral blood cells:

• Pre-treatment with GCSF prior to collection

• G-PBMNCs collected

Cell-based immunotherapy

• Allogeneic T cells (from same donor) transfected with HSV-TK – Zalmoxis approved in 08/16

  • no longer authorised

• Engrafts and helps reconstitute the immune system alongside HSCT from same donor, improves overall survival and non-relapse mortality

• Ganciclovir administered if graft versus host disease develops

  • when too much t-cell produced

  • ganciclovir kills them off

immunotherapy in cancer

• Cytotoxic T cells (T_c cells) are activated by antigen presenting cells such as dendritic cells, which prime them with specific antigens for target cells (e.g. infected cells, cancers)

• presented on surface via MHC

• T_c cells then recognize and kill cells which express these antigens via release of perforin and granzyme, which induce apoptosis

The efficacy of this process can be massively improved using cell engineering approaches

APCs

Antigen Presenting Cell

• used to train t-cells to respond to the cancerous antigen

• Artificial APCs are currently an emerging approach – decorate cells or microparticles with antigens in order to prime T cells

• “Off the shelf” - does not require harvesting from the patient

CAR T-Cells

• T cells genetically engineered to express a chimeric antigen receptor (CAR)

• CAR T cells proliferate and kill tumour cells upon contact with the antigen they recognize

Kymriah

• first ever FDA approved CAR-T cell therapy

• for b-cell lymphocytic leukemia in children and young adults

• recognises CD19

IMLYGIC

• HSV-1 engineered to be oncolytic

• kills cancer and doesn’t harm healthy cells

• it attracts cells of immune system to help fight the leukemia

Mesenchymal Stem Cell Therapy

• Ability to home to site of injury

• Anti-inflammatory properties

• Hypoimmunogenic - no immune response so dont need to match to donor

All makes them suitable as therapeutic cells

Pro-tumorgenic effects of MSC

• promote tumor cell ggrowth invasion and metastasis and support tumour fbrovascular network and promote angionesis

• support the tumor-induced vascular network

MSC in cancer patients

• if delivered into patients with cancer it will hone and embed into cancer cell and produce trail ligand

• trail ligand will react with trail receptor on cells and induce apoptosis

Anticancer effects of MSCs

• anti-angiogenesis

• cytokine delivery

• to deliver miRNA

• vehicle for anti-cancer drugs

CRISPR/Cas9

• Clustered Regularly Interspaced Short Palindromic Repeats

• CRISPR-associated (Cas) genes

Targeted CRISPR/Cas9 treatment

• head and neck cancer due to overexpression of SOX2 gene in cancer stem cells which promototes growth and resistance to apoptosis

• specifically knocking down SOX2 in tumour cells using CRISPR/Cas9 should cause cancer regression

• found a 90% inhibition of growth, 90% increase in survival and tumour disappeared in 50% of mice

dCas9

• a mutated cas9 which deactivates nuclease activity and enables it to be used to control gene expression

• guide RNAs localise dCas9 to specific genes where transcription is activated or repressed

CRISPR-Based cell therapies

• trials with engineered t-cells with `

• CRISPR knocks out endogenous T-cell receptor (stops knocking out what it’s linked well) and PD-1 (stops cancer cell from inhibiting t-cells)

• therapy was well-tolerated

• but only 10% of t-cells in trial has all of the genetic modifications needed

Using genome editing to be able to target drug delivery

• looked at cancer patients and looked at the 1000+ variations of p53

• tested variations to see if specific mutations caused cancer to see if there are any biomarkers for cancer or any specific druggable targets

Advantages of gene editing

• Small (potentially easier to deliver), inexpensive, specific, versatile

• Developments in base and prime editing offer more control

Disadvantages of gene editing

• Off-target effects - unintentional editing similar genomic regions to the target, possibly oncogenic

• Autoimmune risks - possible autoimmune responses or unintended effects on healthy tissues

• Efficient delivery to all target cells or a specific subsets remains challenging

• Ethical Concerns

• Limited simultaneous changes - CRISPR currently allows limited simultaneous changes per cell

• HDR to NHEJ ratio - the balance between the two affects editing efficiency

• Abuse, terrorism - people injecting themselves with CRISPR/Cas9