Gene Therapy

Gene therapy

• Aims to produce a therapeutic effect

• Through manipulation of gene expression or altering the biological properties of cells

• The approach is dependent on the goal

• in vivo and ex vivo routes

Cancer treatment

Target tumour cells → express foreign antigen → immune clearance

Monogenic disorders

• Are germline and inherited changes

• Therapy targets somatic cells

• Gene addition - recessive

• Gene replacement - recessive/dominant

• Gene correction - recessive or dominant

Method

1. Select approach - add, edit, silence, correct

2. Selective delivery method and route (get the gene to the target cell)

3. Check appropriate expression in target tissue

in vivo methods

• Introduce modified cells into patient

• Extract stem cells

• Collect donor cells

ex vivo method

Lipid nanoparticle

Considerations of applying gene therapy

• Immunogenicity viral coat proteins

• Gene silencing

• Cell cycle

• Integration

• Gene editing/off-target editing

• Validity of model it was developed in

Immunogenicity viral coat proteins

T-cell response can limit dose or allergies

Cell cycle

Some viruses won’t enter non-dividing cells, so it restricts targeting

Integration

• Non-integrating is soon lost and is a short-term effect

• Integrating is permanent and may cause insertional mutagenesis

Gene silencing

Introduced genes modified by methylation, but it’s a short-term effect

Human embryo testing

• The safety of techniques is unknown as limited no. of people have received these treatments

• Potential for ‘off-target’ effects is larger when considering correcting polygenic disorders

• Approaches are now considering editing germ-line so there needs to be a review of legislation before approved for patient trials

• What is considered ‘correctable’

• How gene therapies should be made available

• Informed consent - unsure what will happen to those affected and their gametes

Adenovirus (Ad)

• DNA

• 7.5kb and vectors can carry up to 30kbp

• Respiratory tract, eyes and liver

• Case study - Liver OTC deficiency

Adenovirus cell cycle, integration, production, problem

• Cell cycle - infects dividing and non-dividing cells

• Integration - no, it’s unlikely to cause insertional mutagenesis and short-lived effects

• Production - easy, gutted vectors require helper phage

• Problem - immune response, no repeat use

Liver OTC deficiency

• Lack of ornithine transcarboxylase in the liver

• Causes a lack of ammonia clearance from blood

• Severe OTC causes death during infancy

• Mild OTC can be helped with dietary control and survival to adulthood

Liver OTC deficiency gene therapy

• Inject Ad carrying OTC gene into hepatic artery

• Targets the liver

• Binds Coxsackie-Adenovirus Receptor (CAR) on the cell surface

• Ad genes expressed within 24 hours

• Crucial if patient goes into coma and can then use liver transplant for long-term treatment

• Tested on mouse model and treatment worked well

Liver OTC deficiency human gene therapy trial

• 18 adult volunteers with mild OTC deficiency given Adenovirus

• 1 died from massive systematic immune response to the adenoviral vector

• Inflammation was not confined to the liver

Liver OTC deficiency review of gene therapy

• Hint of toxicity had cropped up in previous experiments but these were not recognised

• No in-trial assessments

• One crucial aspect in the animal data did not translate to humans - the Coxsackie-Adenovirus Receptor (CAR)

Adeno-associated virus (AAV)

• DNA (almost invisible to the immune system)

• 4.5kb - small size may restrict use

• Site is wide ranging but can restrict using tissue-specific promoters

• Case study - Leber congenital amaurosis (LCA)

• Case study - Duchenne muscular dystrophy

Adeno-associated virus (AAV) cell cycle, integration, production, immunological response

• Cell cycle - infects dividing and non-dividing cells

• Integration - yes, there is long-term expression

• Native virus at specific site at chromosome 19 vector removes ‘int’ gene causing random integration

• Production is difficult

• No associated immunological response

• Can be contaminated with adenovirus or herpes virus

Leber congenital amaurosis (LCA)

• 20 genetic causes both AR/AD inheritance

• Congenital retinal degeneration - RPE65 (AR)

• Fail to make a functional photoreceptor, causing retinal degeneration

Leber congenital amaurosis (LCA) gene therapy option

• Inject AAV

• Expressing REP65 beneath the retina

• Delivers a functional copy of RPE65 in RPE to compensate LOF

• Tested in mice and canine models

Leber congenital amaurosis (LCA) human gene therapy trial

• Assay for pupil dilation in response to light to each eye alternatively

• Big improvement and no documented side-effects

Duchenne Muscular Dystrophy

• Non-functional dystrophin with muscle degeneration

• Antisense oligonucleotide can facilitate exon skipping for patients with specific variants

Duchenne Muscular Dystrophy gene therapy

• Age 4-5 years

• No AAV rh74 Ab

• Elevidys is a recombinant gene therapy

• Delivered in an AAV that results in production of a micro-dystrophin containing key domains

• Single IV dose

Duchenne Muscular Dystrophy human gene therapy trial

• No clear benefit seen in clinical outcome

• No safety concerns either, so it progressed

Retroviruses

• RNA (invisible to immune system)

• 8kb, replace pathogenic genes

• Infection is wide ranging

• Low efficiency in vivo vs. high efficiency ex vivo

• Case study - X-linked severe combined immunodeficiency

Retroviruses cell cycle, integration and production

• Cell cycle - dividing cells only which is restrictive

• Integration - yes, long-term expression with random integration and insertional mutagenesis

• Production is difficult

• Early promise, but some setbacks

X-linked severe combined immunodeficiency

• Prone to recurrent and persistent infection

• Life expectancy under 2 years - must be confined to sterile bubble prior to bone marrow transplant

• X-SCID - yc cytokine receptor (deficient in T-cells and NK cells)

• ADA-SCID - adenosine deaminase (deficient in T-cells)

X-linked severe combined immunodeficiency animal model

• yc -/- mice are very immunodeficient (a number of SCID mouse models)

• The phenotype can be rescued by ex vivo transfection of hematopoietic stem cells with retrovirus

• Transfected cells are selected and expanded within immune system

X-linked severe combined immunodeficiency gene therapy option

• Only needs to integrate into a few cells to expand T and NK population

• Very efficient

• Gene expression silenced in some cells over time

• System selects and expands cells with continued expression - long term solution

X-linked severe combined immunodeficiency human gene therapy trial

• Treated 11 children with a retrovirus encoding y/c cytokine receptor

• 10/11 gained remission from the disease

• 5/10 developed leukaemia

• In 2 of these cases, retrovirus integrated near the oncogene Imo2

Lentiviruses

• RNA (invisible to immune system)

• 8kb capacity and replaces pathogenic genes

• Site is wide ranging where it expresses targeting proteins in viral coat

• Case study - X-linked adrenoleukodystrophy (ALD)

Lentiviruses cell cycle, integration, production, problems

• Cell cycle - infects dividing and non-dividing cells

• Integration - yes, long-term expression and random integration problems

• Production is extremely difficult

• Problems - slow response, production, integration

X-linked adrenoleukodystrophy (ALD)

• Variants in ABCD1 peroxisomal transporter are fatal in childhood

• ABCD1 required for turnover of myelin

• Demyelination results in neurodegeneration

• Treatment - allogenic bone marrow transplant

• Donor HSCs arrest disease progression

• Migrate to recipients CNS replacing microglia

X-linked adrenoleukodystrophy (ALD) human gene therapy trial

• 2 patients

• HSCs corrected ex vivo with HIVlentivirus ABCD1 gene

• 9% and 14% of bone marrow progenitors express ABCD1 after 30 months

• Cerebral demyelination arrested after 14-16 months

• Cognitive functions stabilised

• Outcome similar to successful bone marrow transplant

• HSC were polyclonal for ABCD1 - no favoured site of integration

• Success was limited to those diagnosed early with limited disease symptoms

Methods for genome editing delivering systems

• Considered several biological methods – viral vector systems

• Chemical methods

• Physical methods

Chemical methods

• Nanoparticles

• Lipoparticles

Physical methods

• Electroporation

• Sonoportation

• Infusion techniques

• Microinjection

CRISPR/Cas

• TDNA - wide ranging and PAM specificities apply

• Insertion and deletion changes to knock-out a gene

• Alter gene expression

• Design and production is simple

• Case study - MYPBC3

CRISPR/Cas capabilities

• Elevate or depress gene expression by targeting tissue specific enhancers

• Use an inactive Cas9 fused to a transcriptional activator or repressor domain

• Good for ‘haploinsufficiency’ disorders

• Target splice acceptor and donor sites

MYBPC3

• Hypertrophic cardiomyopathy disease

• Heart muscle becomes thickened

MYBPC3 gene therapy

• AD variant in MYBPC3 affects muscle structure in the heart

• Use CRISPR/Cas9 and DNA repair template

MYBPC3 embryo gene therapy

• Adding CRISPR-Cas9 to the fertilised egg

• 20% success rate but high levels of mosaicism

• Add CRISPR-Cas9 along with sperm

• 72% embryos show correction and only 1/42 is mosaic

• IVF (PGD) should remain the standard of care

MYBPC3 embryo gene therapy concerns

• Mosaicism

• Off-target editing

• Detection of abnormalities in edited embyros

MYBPC3 human gene therapy

• Heavily criticised work

• Mosaicism

• Potential off-target site

• Germline edit

Base-editing therapies

• Cytidine deaminase converts C → U, which is read as T

• Adenine deaminase converts A → G

• Modified Cas9 acts as a ‘dual-nickase’ - cuts one strand at a time to promote HDR

• DNA - wide ranging with PAM specificities apply

• Indel and point changes

• Design and production are simple

• Case study - T-cell acute lymphoblastic leukaemia

• Case study - PSK9

Base-editing therapies problems

• Too early to know

• Immunological response and editing off-target locations are unknown

T-cell acute lymphoblastic leukaemia

• Goal - cure all in a patient that exhausted conventional treatment options

• Previously treated with chemotherapy and bone marrow transplant and disease reoccurred

T-cell acute lymphoblastic leukaemia gene therapy

• Donor CAR T-cells were modified

• Reprogrammed to eliminate cancerous T-cells

• Protected from the patient’s immune system

• All achieved by base editing

T-cell acute lymphoblastic leukaemia human gene therapy

• Within 28 days, patient in remission

• Received a second bone marrow transplant to restore immune system

• Clinical trial opens to 10 more patients

PSK9

• PSK9 controls levels of low-density lipoprotein

• High levels cause HCL

• Goal - reduced PSKC9

PSK9 gene therapy

• VERVE-101 is an adenine base editor

• Delivered in lipid nanoparticle by injection

• Taken up by liver

• Targets A to G

• Prematurely truncate PCSK9

PSK9 human gene therapy

• VERVE-101 reduced amount of LDL by 55% in 6 months

• Up to 84% drop in PCSK9 levels

• 2/10 participants had life-threatening cardiac events

Sickle cell anaemia

• Point variation in beta-globin gene

• Causes glutamic acid to change to valine

• Causes RBCs to sickle under hypoxic conditions

Sickle cell anaemia - changing strategy over time

1. Allogenic bone marrow transplantation

2. Gene addition/silencing

3. Gene editing with CRISPR/Cas9

Allogenic bone marrow transplantation

Affected cells are derived from haemopoietic stem cells

Gene addition/silencing

• Addition haemoglobin gene to stem cells

• Silence BCLA11A gene known down

• Elevate HbF levels

Gene editing with CRISPR/Cas9

1. Correct beta-globin mutation with donor DNA template

2. Truncate BCLA11A transcription factor (negative regulator of HbF)

Problems with standard viral vector approach

• Length of DNA that can be packaged

• If non-integrated, it may soon be lost

• If integrated, it may cause insertional mutagenesis

Problems with gene-editing approach

• Can’t model structural changes

• Off-target editing unpredictable

• Misuse and germline changes

Cystic fibrosis

• Monogenic disorder

• Point mutation in CFTR gene

• Autosomal recessive

• 1 in 24 Europeans are carriers (heterozygotes)

• Current treatment directed at symptoms rather than the cause

• Life expectancy is ~30+ years