Inherited metabolic diseases (IMD) are single gene disorders caused by enzymatic defects in metabolic pathways in which cumulative incidence is estimated as high as 1/800 (1, 2). Standard of care may include diet, enzyme and coenzyme replacement, removal of harmful substances, cell and organ transplantation and supportive therapies (3). In the past 20 years, gene therapy has emerged as a disease-changing treatment for these disorders (4).
Gene therapy is simple in principle, restoring normal cellular function by providing a functional copy of the defective gene by the addition of a new copy of the gene or tools to ‘edit’ the defective gene and correct the genetic mutation with specific vectors (5). Gene therapy had driven fantastic hope in the mid-1990s when addressing severe combined immunodeficiency (SCID) due to deficiency of the enzyme adenosine deaminase (ADA-SCID) (6). However, contemporary dramatic adverse events in historical clinical trials have tempered this enthusiasm when an ornithine transcarbamylase-deficient young adult died after a severe immune reaction against the gene therapy vector in a clinical trial (7) and leukaemias developed secondary to insertional mutagenesis in patients with X-linked SCID (X-SCID) (8). Subsequently, the research focused on safer delivery vectors and successful results have currently been reported for various inherited rare diseases such as Leber’s congenital amaurosis (9), X-linked adrenoleukodystrophy (10), metachromatic leukodystrophy (11), haemophilia B (12) and many other IMDs, leading to first market authorisations in Europe and in the USA in 2012 and 2017, respectively (13).
The strategies and delivery technologies developed to improve the efficacy and the safety of gene therapy are a field of intense research and interest. This review will outline the progress made from early stages to ongoing clinical trials by highlighting general principles and emphasising different delivery methods.
Gene therapy is the transfer or editing of a genetic material to cure a disease. Depending on the delivery strategy chosen, gene therapy can be performed
The gene therapy field was recently revolutionised by the introduction of genome editing tools, which includes nucleases engineered to modify the genome at precise loci. This includes zinc finger nucleases (ZFNs) (19), transcription activator-like effector nucleases (TALENs) (20), homing endonucleases (meganucleases) (16) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/(CRISPR-associated system) Cas9 (16). These nucleases generate double-strand breaks in DNA, which is subsequently resolved by cellular DNA repair pathways. Two of these repair pathways are often exploited to mediate gene correction, addition and deletion or disruption: Non-Homologous End Joining (NHEJ) involves direct ligation of two DNA termini with intervening small sequence insertions and deletions (InDels), and Homology Directed Repair (HDR) achieves precise modification of the DNA through the introduction of a DSB along with an exogenous repair template. CRISPR/Cas9 is emerging as a popular tool for both
A massive obstacle to the translation of gene therapy is the high prevalence of immune response against the vectors, either pre-existing (25) when the patient has been previously exposed to the wild-type viral serotype used as gene therapy vector, or after exposure to the vector in the case of a re-injection is required. Viral vectors can evoke an innate immune response via several pathways, such as the sensing of pathogen-associated molecular patterns on vector particles or in the vector genome (26). While adenovirus (Ad) vectors provoke a robust innate immune response via complement activation, and both Toll-like receptor (TLR) TLR-dependent and TLR-independent mechanisms, lentiviral vectors cause an increase in the expression of several cytokines such as interferon (IFN)-a and IFN-β. Immune response against AAV vectors is known to be TLR3-independent, while humoral responses against the AAV capsid are enhanced by the presence of the complement (27). The recognition of these immune responses is not always identified during preclinical safety studies (4). Several approaches have been proposed to overcome this immune response: reducing the vector dose, capsid modifications, tissue-targeted gene transfer and immunomodulation (27).
In early onset IMDs, the deficient enzyme is often associated with a truncated native protein. The expression of the whole functional enzymatic protein can trigger an immune reaction against the transgenic protein itself. This has not been observed so far but remains a theoretical risk.
In most IMDs, an early treatment is essential to prevent debilitating complications, long-term sequelae and potentially death. An early administration can target more progenitor cells, which could theoretically enable a better effect for a same dose of gene therapy vector and can take advantage of an immature immune system. However, in rapidly growing organs such as the liver, non-integrative strategies of gene delivery means a progressive loss of the delivered transgene that will require re-injections, often precluded by a strong immune response triggered by the first injection (4).
Earlier, time for development of a promising gene therapy product—from the first patient injected in a clinical trial to market authorisation—has been slow from 8 to 16 years, although it is expected that the next products to be authorised might be developed at a quicker pace at present that the process has been successfully optimised with the first pioneering products (28).
Various strategies to efficiently deliver nucleic acids to target organs have been developed over the last 30 years and are summarised with their respective advantages and pitfalls (Table 1).
Main features of gene therapy strategies.
Non viral | Lentivirus | Adenovirus | Adeno-associated virus | |
---|---|---|---|---|
No | Yes | Yes | Yes | |
No limit | 14kb | 7.5kb | 4.7kb | |
No | Yes | No | Rarely | |
No | Yes | Yes | Yes | |
No | Insertional mutagenesis | Immune response | Limited immune response |
Kb : kilobases
Lentiviral vectors, such as those based on human immunodeficiency virus type 1 (HIV-1), are a class of retrovirus that are used widely in gene therapy. Lentiviral vectors reverse-transcribe their single-stranded RNA genome on cell entry to form a double-stranded DNA product that translocates to the host nucleus and integrates into the host genome (Figure 1). Controversies over use of retroviral vectors have existed since induction of leukaemia in patients treated for X-SCID via
Transduction pathways of lentiviral and adeno-associated viral (AAV) vectors : cellular uptake and in-cell processing.
In recent years,
Adenoviruses are non-enveloped double-stranded DNA viruses with a large 36 kb genome. They can target both dividing and non-dividing cells. Adenoviral serotypes are triggering a strong innate immune response (4). Adenoviral vectors are non-integrating vectors and their payload remains as circular DNA in the cellular nucleus. These vectors enable long-term expression and can accommodate large transgenes. The strong immunogenicity generated by capsid proteins was associated with a dramatic event in a clinical trial for OTC deficiency when a young patient died following a fulminant immune response and subsequent multiorgan failure (7). Other vectors with improved safety profile have been generated known as helper-dependent adenoviral vectors (HD-Ad) with deletion of most of the coding sequences from the adenoviral genome. The improved safety profile has shown benefit in preclinical studies and in clinical trials, but the acute innate immune response persists. This limits applications in IMD but is an advantage for its use in the field of cancer or vaccination (4).
AAV is a non-enveloped, single-stranded DNA parvovirus with a 4.7 kb genome, which is dependent on co-infection with a helper virus (like adenovirus) to replicate and generate a viral infection (37). It is composed of the
Depending on serotypes, 20–80% of the general population has pre-existing antibodies against the capsid proteins, which is one of the major hurdles for clinical translation. These antibodies, even at low titres, are often neutralising the transduction of the target cell, especially if the vector is delivered in the bloodstream. As there is currently no validated immunosuppressive protocol, which has shown reasonable efficacy in mitigating this risk, clinical trials are recruiting seronegative patients for the AAV capsid used (25, 39).
Due to the small size of the genome and simple viral life cycle, AAV is emerging as one of the most successful gene therapy vectors, particularly with their safety and effectiveness in some monogenic disease trials (40). Several successful trials have been conducted for a variety of inherited diseases since the mid-2000s leading to the first gene therapy product receiving market authorisation in the West for lipoprotein lipase deficiency in 2012 (13).
Liposomes are spherical vesicles enclosing an aqueous space within a synthetic lipid bilayer membrane used as non-viral gene therapy vectors (41, 42). Phospholipids and sphingolipids are commonly used lipids that allow their natural self-assembly (43). Their size varies from 20 nm in diameter to a few microns. They are particularly attractive gene therapy vector carriers as they can transport large pieces of DNA and protect genetic cargo from degradation. Cationic liposomes are more popular as they offer almost 100% loading efficiency. The negative charges in DNA interact with cationic liposomes to form complexes that allow increased DNA uptake (44, 45, 46). They are biocompatible and have low immunogenic risk and no replication risk (47). Their physiochemical and biophysical properties can be modified for drug loading and targeting to specific cells and tissues, such as they have also been used successfully to deliver genes to cells both
Their main challenges are a limited delivery with non-specific binding, reduced stability
Exosomes are small vesicles (30–150 nm in size) naturally secreted by most cell types at the end of the endocytic pathway post-fusion of late endosomes/multivesicular bodies (MVBs) with the plasma membrane (56, 57) and represent another form of non-viral gene therapy vectors. Exosomes play a critical role in cell–cell communication by delivering functional proteins and genetic contents such as mRNA and miRNA transcripts to the recipient cells (58, 59). Hence, they influence both physiological and pathological processes in cells (60, 61).
Due to their natural ability to transfer genetic information, exosomes present themselves as an attractive target to be exploited for therapeutic application (62, 63, 64). They are biocompatible and are composed of non-viral components that reduce their immunogenic risk and ensure their longevity in circulation. Their bilayered lipid structure can protect the genetic cargo from degradation, and their small size and flexibility can facilitate them to cross major biological membranes including the blood–brain barrier (65, 66). They can be modified to enhance their targeting abilities to specific cells and tissues (67). The main challenges include robust and reproducible methods for exosomes manufacturing, characterisation and efficient cargo loading (68), immunogenicity (69) and a limited understanding of exosome biology (57, 70).
However, encouragingly, preclinical studies have shown the safety of multiple injections (71). While the use of exosomes in IMD remains at its early days, recent studies used engineered exosomes loaded with b-glucocerebrosidase (GBA)
The use of messenger RNA (mRNA) delivery for gene transfer is an appealing strategy as mRNA can be translated rapidly into protein once reaching the cytoplasm. mRNA provides temporary, half-life-dependent protein expression that allows adjustable protein production.
mRNA is often delivered via lipid nanoparticles (74, 75), a strategy explored in few IMDs (76, 77). LNPs are liposome-like structures encapsulating genetic materials like RNA and DNA but are made of single lipid layer resulting in solid/lipophilic core (78, 79). They can assume micelle-like structures to encapsulate drugs in their non-aqueous core, have high delivery rates and better endosomal escape and show low lipid accumulation in target organs. Lipid nanoparticles are composed of phospholipids, sterols and polyethylene glycol (80)-conjugated lipids, which protect the mRNA from nuclease degradation and immune responses and help in their cellular uptake (81). mRNAs encapsulated in LNPs have been trialled in animal models of methylmalonic acidaemia (82), acute intermittent porphyria (83), ornithine transcarbamylase (84) deficiency (85), arginase deficiency (86) and galactosaemia (87). Phase I/II clinical trials have been initiated in OTC patients to test the safety and efficacy of OTC mRNA formulated in lipid nanoparticles (76). These advancements in liposome-based cargo carriers will undoubtedly provide a platform in further development of gene therapy non-viral vector carriers.
Proof of concept of gene therapy has been achieved in many animal models recapitulating the human phenotype of IMDs: urea cycle defects, organic acidurias, maple syrup urine disease, phenylketonuria, tyrosinaemia type 1, glycogen storage disease type Ia, long-chain fatty acid oxidation disorders, homozygous familial hypercholesterolaemia, lipoprotein lipase deficiency, primary hyperoxaluria type I, progressive familial intrahepatic cholestasis, Wilson disease (4), Pompe disease (88), Gaucher disease (89), mucopolysaccharidosis (90, 91) and mitochondrial diseases such as mitochondrial neurogastrointestinal encephalomyopathy (92). For example, AAV vectors have successfully targeted the four most common urea cycle defects: OTC deficiency (93), citrullinaemia 1 (94), argininosuccinic aciduria (95) and arginase deficiency (96). It is noteworthy that the extrapolation of data for dose finding is often reliable from animal studies to clinical trials (28).
While most of these studies are using
Various clinical trials for IMD are currently planned, ongoing or completed, targeting mainly the brain or the liver. A non-exclusive list is provided in Table 2. Most trials are using either
Indicative list of gene therapy clinical trials for inherited metabolic diseases in 2020.
Inherited metabolic diseases |
Disease | Sponsor | Phase | Status | Vector | NCT number ( |
---|---|---|---|---|---|---|
Glycogen storage disease 1A | Ultragenyx | I/II | R | AAV8 | NCT035117085 | |
Crigler-Najjar | Genethon-Selecta Bio | I/II | R | AAV | NCT03466463 | |
Ornithine transcarbamylase deficiency | University of Pennsylvania | I | T | Adeno- viral | NCT00004498 | |
Ultragenyx | I/II | R | AAV8 | NCT02991144 | ||
Intermediary metabolism | Methylmalonic acidaemia | Moderna Therapeutics | I/II | R | Non viral | NCT03810690 |
Propionic acidaemia | Moderna Therapeutics | I/II | A-NR | Non viral | NCT04159103 | |
Phenylketonuria | Homology Medicines | I/II | R | AAVH- SC15 | NCT03952156 | |
National Taiwan University Hospital | I/II | T | AAV2 | NCT01395641 | ||
Aromatic L-amino acid decarboxylase deficiency | National Taiwan University Hospital | II | R | AAV2 | NCT02926066 | |
National Institute of Health | I | R | AAV2 | NCT02852213 | ||
Lipid metabolism | Homozygous Familial |
RegenX Bio | I/II | R | AAV | NCT02651675 |
Sangamo Therapeutics | I/II | H | AAV6 | NCT02702115 | ||
Mucopolysaccharidosis 1 | RegenX Bio | I | R | AAV9 | NCT03580083 | |
Orchard Therapeutics/San Raffaele-Telethon Institute for Gene Therapy | I/II | R | LV | NCT03488394 | ||
Mucopolysaccharidosis 2 | Sangamo Therapeutics | I/II | H | AAV6 | NCT03041324 | |
RegenX Bio | I/II | R | AAV9 | NCT03566043 | ||
Manchester University | I/II | R | LV | NCT04201405 | ||
Lysogene | I/II | T | AAVrh10 | NCT01474343 | ||
Mucopolysaccharidosis 3A | Lysogene | I/II | R | AAVrh10 | NCT03612869 | |
Abeona Therapeutics | I/II | R | AAV9 | NCT02716246; NCT04088734 | ||
Mucopolysaccharidosis 3B | Abeona Therapeutics | I/II | R | AAV9 | NTC03315182 | |
Uniqure | I/II | T | AAV5 | NCT03300453 | ||
Mucopolysaccharidosis 6 | Fondazione Telethon | I/II | R | AAV8 | NCT03173521 | |
Audentes Therapeutics | I/II | A-NR | AAV8 | NCT04174105 | ||
Spark Therapeutics | I/II | A-NR | AAV | NCT04093349 | ||
Lysosomal storage diseases | Pompe disease | Florida University | I/II | T | AAV1 | NCT00976352 |
Florida University | I | R | AAV9 | NCT02240407 | ||
Danon disease | Rocket Pharmaceuticals | I | R | AAV9 | NCT03882437 | |
Sangamo Therapeutics | I/II | R | AAV6 | NCT04046224 | ||
Fabry disease | Freeline Therapeutics | I/II | R | AAV | NCT04040049 | |
AvroBio | I/II | R | LV | NCT03454893 | ||
Ceroide lipofuscinosis 6 | Amicus Therapeutics | I/II | A-NR | AAV9 | NCT02725580 | |
Ceroide lipofuscinosis 3 | Amicus Therapeutics | I/II | R | AAV9 | NCT03770572 | |
Cornell University | I | A-NR | AAV.rh10 | NCT01161576 | ||
Ceroide lipofuscinosis 2 | Cornell University | I | A-NR | AAV2 | NCT00151216 | |
Cornell University | I/II | A-NR | AAV.rh10 | NCT01414985 | ||
GM1 Gangliosidosis | National Human Genome Research Institute | I/II | R | AAV9 | NCT03952637 | |
Metachromatic leukodystrophy | Orchard Therapeutics/San Raffaele-Telethon Institute for Gene Therapy | I/II | R | LV | NTC03392987 | |
Shenzhen University | I/II | R | LV | NCT02559830 | ||
Gaucher type 1 | AvroBio | I/II | R | LV | NCT04145037 | |
Bluebird Bio | II/III | A-NR | LV | NCT01896102 | ||
Peroxisomal disorders | X-linked childhood cerebral adrenoleukodystrophy | Bluebird Bio | III | A-NR | LV | NCT03852498 |
Shenzhen Second People's Hospital | I/II | R | LV | NCT0372755 |
Information gathered from
Information gathered from clinicaltrials.gov accessed on 22 January 2020.
A-NR, active-not recruiting; H, on hold; NR, not recruiting; R, recruiting; T, terminated.
X-linked adrenoleukodystrophy is the most common peroxisomal disorder caused by mutations in the
Haemophilia B is a X-linked inherited severe bleeding disorder caused by factor IX (FIX) deficiency with a frequency of 1 in 30,000 male live births. The measurable plasma levels of FIX stratify the clinical severity: severe (<1%), moderate (1–5%) and mild (5–40%). The standard of care relies on injections of recombinant FIX, an expensive therapy for public healthcare systems with an annual cost of US$300,000 (102). As a minimal increase of plasma FIX can dramatically improve the clinical phenotype, various programmes have targeted haemophilia B. A phase I/II trial sponsored by Sparks Therapeutics and Pfizer (NCT02484092) has used an AAV vector expressing an improved variant of FIX cDNA known as Padua variant with a gain-of-function activity enabling a 5–10 times increase of FIX activity levels under the transcriptional expression of the liver-specific hAAT promoter. This vector was administered by a single peripheral intravenous injection at a dose of 5 ´ 1011 vector genomes per kilogram body weight. Preliminary results showed an increase in FIX from <1% at baseline at 1 week after infusion, reaching a plateau at 3 months post-injection and remaining stable over 1 year with a mean sustained FIX activity at 34 ± 18.5%. The annual bleeding rate was significantly reduced (from 11.1 to 0.4 events per year;
Glybera is an AAV1-based vector carrying the lipoprotein lipase transgene, which has shown a reduction in acute pancreatitis episodes in patients with lipoprotein lipase deficiency. This product commercialised by Uniqure was the first gene therapy product approved by the European Medicine Agency (EMA) in 2012. Priced at US$1.1 million per patient, this treatment was administered only to one patient and Uniqure decided to withdraw the product from the market in 2017 due to economic considerations (38). Strimvelis is an
Gene therapies have revolutionised the last half century from science fiction to the first commercialised products. New delivery strategies are constantly in development or refinement to achieve the most efficient and safest approach enabling with a single injection of gene therapy vector, a lifelong cure for severe IMDs. This novel therapy option will find its place among other available therapies, alone or in combination if necessary. Many open questions remain, especially about building an adequate economic model to enable affordable gene therapy products for healthcare systems and the long-term efficacy and safety of these novel therapies. Currently, research is designing novel genotype-specific molecular therapies that aim to become patient-specific and personalised gene therapy approaches to correct the phenotype of genetic diseases more efficiently.