Mednotes

L10 Genetics Gene therapy

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tagsgenetics, lecture
aliasesL10 Genetics Gene therapy

16 min read

Overview

This lecture moves from diagnosing and predicting the course of monogenic disease to the range of ways such disease can be treated, then works through gene therapy specifically: what it is, what a vector needs to deliver it, and how delivery route and promoter choice determine where a gene is expressed. It then applies these principles to three worked case studies, severe combined immunodeficiency (SCID), haemophilia A, and Batten disease, tracing each from molecular defect through to an approved or trialled gene therapy product, before placing these in the context of the wider gene therapy trial landscape.

Diagnosis and prognosis of genetic disease

  • Newborn screening (NZ): only offered where a treatment exists. Disorders screened include amino acid disorders (phenylketonuria, maple syrup urine disease), fatty acid oxidation disorders (e.g. MCAD), congenital hypothyroidism, cystic fibrosis, congenital adrenal hyperplasia, galactosaemia, biotinidase deficiency, and SCID.
  • Genetic testing for rare presentations proceeds through: pedigree analysis (is it likely genetic?), SNP arrays and gene panels, exome sequencing, and genome sequencing (the latter two termed “gene hunters” for their cost), supported by clinical-researcher networks such as GeneMatcher, asking what the mutant gene normally does and whether a drug already targets that mechanism. > [!warning] The gene/pathway labels in the small network diagram illustrating this “gene hunters” process were too small to transcribe reliably.
  • Prognosis depends on the specific mutation; family history can indicate likely course; other genes (modifiers) or environment can alter presentation; prognosis also needs to consider risk to future or current children; rarer diseases often need specialist (sometimes overseas) consultation.

Approaches to treating genetic disease

Three broad strategies: germ-line (gene) therapy, somatic cell gene therapy, and protein replacement therapy (including antisense oligonucleotides, ASOs).

Molecular treatment strategies, each mapped to a step in the gene-to-protein pathway, with a disease example:

  • DNA hypomethylation therapy (e.g. decitabine): increases HbF synthesis in sickle cell disease.
  • Increasing expression from the wild-type locus, or from a mutant locus with residual function: danazol in hereditary angioedema.
  • Gene transfer therapy: SCID (X-linked SCID, ADA deficiency).
  • RNA interference, reducing abundance of an mRNA encoding a dominant mutant protein: corrected clinical defects in a mouse model of spinocerebellar atrophy.
  • Aminoglycoside antibiotics, causing skipping of a premature stop codon so a full-length protein is made: normalised the potential difference of nasal epithelium in cystic fibrosis patients carrying stop-codon mutations.
  • Interfering with a chaperone that traps a mutant protein in the ER: inhibiting a Ca2+-dependent chaperone in ΔF508 cystic fibrosis cells allowed the mutant protein to reach the cell surface.
  • Replacement with a modified protein: Factor VIII or IX in haemophilia; enzyme replacement therapy in Gaucher and Fabry disease.
  • Cofactor therapy, increasing the function/stability of a mutant protein: pyridoxine in classical homocystinuria.

Recap on protein replacement therapy limitations (haemophilia): requires multiple infusions (3x weekly); risk of immune responses to the exogenous protein (inhibitor issues); high cost; possible contaminants in plasma-derived concentrates; recombinant protein is now the standard.

Gene therapy: concept and requirements

Gene therapy treats disease with genetic material, aiming to:

  • replace a mutated gene, by gene editing or by augmentation (adding a functional copy);
  • inhibit expression of a deleterious protein (e.g. Huntington’s disease);
  • kill (cancer) or protect (degenerative conditions) cells.

Main methods are non-viral (DNA or RNA, e.g. ASOs) or viral vectors; administration can be ex vivo or in vivo; expression is regulated through choice of promoter, giving constitutive or inducible expression.

History: concept proposed 1970 (French Anderson); first human treated 1990; 1999 marked on the timeline with a photo of Jesse Gelsinger; first commercial product 2013. A modern single treatment example given: 1.2 million people treated in the US via 60 intramuscular injections in the legs, at $1.2 million.

The four “magic bullet” requirements for a genetic disease therapy: (1) knowledge of the biology of the disease being treated; (2) natural history as a baseline for measuring efficacy; (3) an appropriate delivery method; (4) a means of assessing efficacy.

Viral vectors for gene delivery

No vector is perfect: each trades off packaging capacity for genetic material, tissue tropism, spread, and immune response.

Three vector types and their target-cell binding:

  • Adenovirus 5: surface fibre knob, fibre, fibre shaft, and penton base bind the target-cell receptors CAR and integrin.
  • AAV2: binds the surface receptors HSPG and integrin.
  • Retrovirus/lentivirus: envelope glycoprotein, capsid and envelope bind a cell-surface receptor.

Retro/lentiviral vector genome construct, in order: 5’LTR, ψ (psi packaging signal), Gag/Promoter/Pol, [promoter and gene of interest], Env, 3’LTR. The assembled viral particle carries Gag proteins (MA, CA, NC), Pro-Pol proteins (PR, IN, RT), Env proteins (TM, SU), and RNA at its core.

Promoter choice restricts expression independent of vector spread: rAAV-MLC-2v-GFP was injected intravenously into a young rat; the virus reached heart, liver and kidney, but GFP (visualised by fluorescence at 4 weeks) was expressed only in the heart, because MLC-2v is a muscle-specific promoter.

Delivery strategies: direct vs cell-based

  • Direct delivery: the therapeutic gene is packaged into a vector (e.g. retrovirus) and injected into the patient, reaching a target organ such as the liver directly.
  • Cell-based delivery: the therapeutic gene is combined with cells outside the body, then the modified cells are reintroduced into the patient. Cell sources include genetically modified embryonic stem (ES) cells (which can be engineered to block immune rejection), an ES cell HLA bank, somatic cell nuclear transfer (SCNT)-derived ES cells, adult stem cells isolated and propagated in the lab, or in vitro differentiated stem cells. The gene is packaged into a vector (e.g. retrovirus) and introduced into these cells before reintroduction.

Case study: SCID gene therapy

SCID (severe combined immunodeficiency), “bubble boy disease”, has several forms; the X-linked form is caused by mutation in IL2R, required for normal lymphocyte maturation. David Vetter, diagnosed with SCID, lived 12 years in a germ-free plastic bubble and died after receiving a bone marrow transplant from his sister that contained EBV.

Haematopoiesis: pluripotent stem cells in bone marrow give rise to the lymphocyte series (T cells: help, suppression, cytotoxicity, delayed hypersensitivity, memory; B cells: antibody production; NK cells: killing) and the myeloid series (monocytes and neutrophils: phagocytosis/killing; eosinophils and basophils: antiparasitic/immediate hypersensitivity; platelets: clotting; erythrocytes: O2/CO2 transport). SCID’s IL2R defect affects lymphocyte maturation, so the lymphocyte branch of this lineage is the one disrupted.

SCID today: GlaxoSmithKline applied for European medicines approval for patients without an HLA-matched donor; the first patient was treated with the approved medicine in May 2017.

St Jude gene therapy protocol for X-linked SCID (5 steps): (1) bone marrow harvest from the patient; (2) lentiviral vector transduction, delivering a normal copy of the XSCID gene into the harvested bone marrow stem cells; (3) cryopreservation of the treated marrow until needed; (4) busulfan conditioning, a personalised dose given to the patient to make room for the treated marrow; (5) cell infusion, returning the treated bone marrow stem cells to the patient.

Case study: haemophilia gene therapy

Haemophilia is considered a good gene therapy target because: the molecular defect is well defined; it is a loss-of-function defect suited to gene addition/augmentation; it is recessive; the liver is a readily accessible target tissue; the F8 cDNA (~9 kb) can be reduced in size while retaining function, allowing it to fit into (some) viral vectors; precise control of protein level is not required, since Factor VIII is itself regulated at the protein level via thrombin; and it converts a requirement for infusions 3x/week into a one-time treatment.

Phase III trial of valoctocogene roxaparvovec (vector AAV5-F8, modified to fit the small AAV package): enrolled 134 adult men (over 18) with severe haemophilia A (Factor VIII activity ≤1 IU/dl); excluded patients with AAV5 antibodies, F8 inhibitor, or liver disease.

  • Factor VIII activity: mean increase of 41.9 IU/dl (95% CI 34.1-49.7, P<0.001) over the 52-week study.
  • Treated bleeding episodes: annualised rate fell by a mean of 4.1 events/yr, from a baseline of 2.8 (median)/4.8 (mean, 95% CI) to about 0.8 after infusion, an 83.8% reduction.
  • Factor VIII infusions: annualised rate fell by a mean of 133.9 infusions/yr, from a baseline of 128.6/135.9 to about 2.0 after infusion, a 98.6% reduction.
  • Side effects: liver inflammation and a drop in Factor VIII activity after 1 year in some patients (“not perfect”); 16.4% of patients had serious adverse events.
  • FDA approved June 2023 as Hemgenix.

Landscape of gene therapy trials and approved products

Current gene therapy clinical trials by indication (FDA data): cancer diseases 66.6%, monogenetic diseases 11.5%, infectious diseases 6.3%, cardiovascular diseases 6.2%, other diseases 2.2%, healthy volunteers 1.9%, neurological diseases 1.8%, gene marking 1.7%, ocular diseases 1.3%, inflammatory diseases 0.5%.

42 (and counting) FDA-approved cell and gene therapies, split into:

  • In vivo gene therapy drugs (applied directly to the patient): Gendicine (Tp53), Neovasculgen (VEGF), Glybera (LPLS447X gene), Luxturna (hRPE65 gene), Vitravene (ASO, CMV retinitis), Spinraza (ASO, SMN2 pre-mRNA), Onpattro (RNAi, transthyretin gene), Kynamro (ASO, apolipoprotein B-100), Imlygic (HSV-1 oncolytic virus, GM-CSF gene), Eteplirsen (morpholino oligomer, exon 51, DMD), Oncorine (E1B 55kDa mutant adenovirus, dl1520), Defitelio (single-stranded oligonucleotides, VOD), Macugen (RNA oligonucleotide, VEGF165 isoform), Rexin-G (retroviral vector encoding a cyclin G1 inhibitor), Zolgensma (onasemnogene abeparvovec).
  • Ex vivo gene therapy products (cells modified outside the body, from a donor or the patient): allogenic T cells, Zalmoxis (suicide HSV-TK-ΔLNGFR gene); allogenic chondrocytes, Invossa (TGFβ1 gene); autologous CAR T cells, Yescarta (anti-CD19-CD28-CD3zeta CAR) and Kymriah (anti-CD19-CD137-CD3zeta CAR); autologous haematopoietic stem cells, Strimvelis (ADA gene).

Case study: Batten disease

Batten disease is a group of 13 inherited lysosomal storage diseases of childhood, affecting up to 1:12,500. A case example (Joe, followed from age 4 to 7) illustrates the progressive course: inability to walk, talk, swallow or feed develops, alongside blindness, motor problems, learning difficulties, hallucinations and seizures.

CLN2 Batten disease, protein (enzyme) therapy: the enzyme is too large to cross the blood-brain barrier systemically, so it needs direct infusion; because of its stability, this means 4-hour infusions via a cannula into the brain once every 2 weeks (chest ports are now also being trialled). This successfully delays disease onset but carries risks, and is now being used pre-symptomatically. Approved as a drug (Brineura) in the US, Europe and Australia, at $750,000 US/year.

Slide 23 embeds what appears to be a family testimonial video (Ollie and Amelia, who have Batten CLN2 disease); the rendered image showed only a solid black rectangle, so its content could not be transcribed.

CLN5 gene therapy in sheep (NZ research): CLN5-/- sheep received an intracranial injection of vector (LV, ssAAV9, or scAAV9.CLN5). Disease severity was tracked with the oBDRS score (0-40) against age in months across five groups: control sheep (n=7) stayed flat near 40; untreated sheep (n=7) declined steeply, from about 38 to about 17 by around 19 months; the three treated groups (LV at 2 months, n=3; ssAAV9 at 3 months, n=3; scAAV9 at 7 months, n=4) all declined more gradually than the untreated group, tracking above it, with the scAAV9 group ending lowest of the treated groups (around 27-28 by about 31 months). Source: Mitchell et al., Molecular Therapy 2018.

Human gene therapy for CLN5: development relied on natural history studies (how the disease would progress untreated) and on testing the human clinical vector in animal models for safety, toxicity and efficacy. FDA orphan drug designation was awarded in 2020, giving exclusive development rights for CLN5 gene therapy. The first clinical trial recruited and treated 6 patients; financing issues have prevented recruiting further patients at this point, but the current patients continue to be monitored.

Human gene therapy for Batten disease more broadly is moving forward, including personalised approaches for rare NZ cases, involving groups such as Sanford Research, Nationwide Children’s, and the Charlotte + Gwenyth Gray Foundation. > [!warning] The newspaper-clipping text shown on slide 27 was too small/low-resolution to transcribe reliably.

Key points

  • Recombinant protein sources are key to treatment, removing reliance on tissue-derived (donor) supplies.
  • Gene therapy should eventually become clinically viable more broadly, and is already possible in haemophilia, SCID, and rare fatal diseases such as Batten disease.

Self-test

  1. List the four methods used to reach a genetic diagnosis in a patient with a rare disease presentation.
  2. Besides the specific mutation itself, name two factors that can influence the prognosis of a genetic disease.
  3. Distinguish germ-line gene therapy, somatic cell gene therapy, and protein replacement therapy as approaches to treating genetic disease.
  4. Describe the RNA interference-based molecular treatment strategy discussed, and its disease example.
  5. Explain how aminoglycoside antibiotics can benefit some cystic fibrosis patients, and what effect this produced when tested in nasal epithelium.
  6. List three limitations of protein replacement therapy for haemophilia.
  7. Define gene therapy and list its three broad therapeutic goals.
  8. List the four “magic bullet” requirements needed for a successful gene therapy for genetic disease.
  9. Name the surface receptors bound by adenovirus 5, AAV2, and lentiviral vectors respectively.
  10. Explain why GFP was expressed in the heart but not the kidney or liver after intravenous injection of rAAV-MLC-2v-GFP.
  11. Distinguish direct gene delivery from cell-based gene delivery, and list the possible cell sources for cell-based delivery.
  12. Describe the five steps of the St Jude gene therapy protocol for X-linked SCID.
  13. Which gene is mutated in X-linked SCID, and which blood cell lineage does this predominantly affect?
  14. List four features that make haemophilia a good target for gene therapy.
  15. Describe the outcomes of the Phase III valoctocogene roxaparvovec (Hemgenix) trial for haemophilia A.
  16. Distinguish an in vivo from an ex vivo gene therapy product, giving one named example of each.
  17. Explain why CLN2 Batten disease requires direct intracranial enzyme infusion, and describe the treatment regimen and its approved product.
  18. Describe the design and outcome of the CLN5 sheep gene therapy study.
  19. Using haemophilia and SCID as examples, describe the features of a monogenic disease that make it a favourable gene therapy target, and explain why a viral vector’s tropism does not by itself determine which tissue expresses the delivered gene.

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