Current Clinical Applications Of In Vivo Gene Therapy With AAVs Part 1
Jul 24, 2024
Hereditary diseases are caused by mutations in genes, and more than 7,000 rare diseases affect over 30 million Americans.
Genetic diseases are diseases caused by gene mutations, such as congenital deafness, brain degeneration, and so on. The occurrence of these diseases is determined by genetic factors, and memory is an important cognitive ability that we must use in our daily lives.
However, genetic diseases do not necessarily hurt memory. Many studies have shown that although there is a certain connection between some genetic diseases and memory, many genetic factors do not have any effect on the memory of some people.
For example, studies have shown that some people may carry the Alzheimer's disease gene, but they may still be able to maintain normal memory and cognitive abilities in their later years. Although these people are at risk of developing Alzheimer's disease, they are not necessarily affected by the disease.
In addition, many genetic factors may have a positive effect on a person's memory. For example, some studies have shown that some people with myopia genes can remember the location of static objects better. These people have special cognitive abilities, which may be related to their myopia genes.
In addition, some people may carry some gene mutations that make it easier for people to remember and see things. These genetic factors give these people more excellent cognitive abilities.
Therefore, although there is a certain connection between hereditary diseases and memory, we should view this relationship positively. Memory is an important cognitive ability, and it can be improved with proper training. Perhaps we can regard hereditary diseases as a challenge for us to improve our memory, overcome difficulties, and enhance our cognitive abilities. It can be seen that we need to improve our memory, and Cistanche can significantly improve our memory because Cistanche is a traditional Chinese medicine with many unique effects, one of which is to improve memory. The efficacy of Cistanche comes from the various active ingredients it contains, including tannic acid, polysaccharides, flavonoid glycosides, etc. These ingredients can promote brain health in many ways.
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For more than 30 years, hundreds of researchers have maintained that genetic modifications would provide effective treatments for many inherited human diseases, offering durable and possibly curative clinical benefits with a single treatment.
This review is limited to gene therapy using adeno-associated virus (AAV) because the gene delivered by this vector does not integrate into the patient genome and has a low immunogenicity.
There are now five treatments approved for commercialization and currently available, i.e., Luxturna, Zolgensma, the two chimeric antigen receptor T cell (CAR-T) therapies (Yescarta and Kymriah), and Strimvelis (the gammaretrovirus approved for adenosine deaminase-severe combined immunodeficiency [ADA-SCID] in Europe). Dozens of other treatments are under clinical trials.
The review article presents a broad overview of the field of therapy by in vivo gene transfer. We review gene therapy for neuromuscular disorders (spinal muscular atrophy [SMA]; Duchenne muscular dystrophy [DMD]; X-linked myotubular myopathy [XLMTM]; and diseases of the central nervous system, including Alzheimer's disease, Parkinson's disease, Canavan disease, aromatic L-amino acid decarboxylase [AADC] deficiency, and giant axonal neuropathy), ocular disorders (Leber congenital amaurosis, age-related macular degeneration [AMD], choroideremia, achromatopsia, retinitis pigmentosa, and X-linked retinoschisis), the bleeding disorder hemophilia, and lysosomal storage disorders.
Hereditary diseases are caused by mutations in genes. There are more than 7,000 rare diseases affecting 30 million Americans, i.e., about 10% of the population. There are several hundred million patients around the world, according to the National Organization for Rare Disorders. Two-thirds of the patients are children. Currently, there are no effective therapies for more than 95 percent of these patients.
The few drug-based treatments approved for genetic diseases at best manage or modify symptoms. However, they do not address the underlying genetic cause of the disease.
Thus, these drugs must be administered for life. Hundreds of researchers have dedicated their lives to the pursuit of what initially appeared as an impossible dream: the development of gene therapies for hereditary diseases, i.e., a one-time curative repair or change to an individual's affected gene that minimizes or even eliminates the symptoms for the entire life of the patient.
This dream is now a reality: gene therapy greatly improves the outlook for currently incurable hereditary diseases! This review is limited to gene therapy using adeno-associated virus (AAV) because the gene delivered by this vector does not integrate into the patient genome.
Glybera was approved by the US Food and Drug Administration (FDA) in October 2012 as the first AAV-mediated gene therapy to reach this milestone. Glybera corrected hereditary lipoprotein lipase deficiency (LPLD), which manifests as pancreatitis, recurrent abdominal pain, and eruptive fat-filled spots that result from very high triglyceride levels.
However, the rarity of the disease (1 per million), the cost to the patient, and the expense to maintain therapeutic readiness by the company made it very difficult to continue gene delivery commercially. This form of gene therapy was no longer made available after 2018, at which time, only 31 people in the world had been treated.
There are now five treatments approved for commercialization and are currently available, i.e., Luxturna, Zolgensma, the two chimeric antigen receptor T cell (CAR-T) therapies (Yescarta and Kymriah), and Strimvelis (the gammaretrovirus approved for adenosine deaminase-severe combined immunodeficiency [ADA-SCID] in Europe). Dozens of other treatments are under clinical trials.
The review article presents a broad overview of the field of therapy by in vivo gene transfer, which is based on the direct administration of a gene therapy vector to the body rather than the transplant of gene-corrected cells.
Herein, we will review in vivo gene therapy for neuromuscular disorders (spinal muscular atrophy [SMA]; Duchenne muscular dystrophy [DMD]; X-linked myotubular myopathy [XLMTM]; and diseases of the central nervous system [CNS], including Alzheimer's disease [AD], Parkinson's disease [PD], Canavan disease [CD], aromatic L-amino acid decarboxylase [AADC] deficiency, and giant axonal neuropathy [GAN]), ocular disorders (Leber congenital amaurosis [LCA], age-related macular degeneration [AMD], choroideremia, achromatopsia [ACHM], retinitis pigmentosa, and X-linked retinoschisis [XLRS]), the bleeding disorder hemophilia, and lysosomal storage disorders (LSDs).
In each of these fields, the progress is fantastic, clinical trials are underway, and in some cases, the treatments are approved by regulatory agencies and commercialized.
Clinical Gene Therapy in Neuromuscular Disorders
Clinical gene therapy in its various forms is rapidly evolving, offering the glimpse of hope that the broader community of rare disorders has long awaited. The most promising viral vector for gene transfer for neuromuscular diseases is AAV, having an excellent safety profile and efficiency in translation.
We herein review three promising clinical AAV gene-therapy approaches that show promise for patients with severe and debilitating neuromuscular diseases.
SMA
SMA is a devastating neurodegenerative disease resulting from progressive loss of motor neurons.1
This autosomal recessive disorder results from a mutation in the survival motor neuron SMN1 gene with an incidence of approximately 1:10,000 live births, 60% of whom have SMA type 1.2,3 The human SMN gene is an inverted duplication on chromosome 5q13.2.
SMN1 is telomeric and the highly homologous SMN2, lying in a centromeric position.1,4 An exon 7-point mutation of the SMN2 gene results in exon splicing and exclusion from the final transcript, resulting in an unstable degradable protein.5
A full-length functional SMN protein is primarily the responsibility of SMN1 with a small contribution from SMN2. 6 In the absence of SMN1, the SMN2 copy number is the major determinant of the clinical phenotype.7
Affected infants with 2 copies of SMN2 are likely to develop severe type 1 (SMA1), characterized by rapidly progressive weakness, inability to sit independently, respiratory insufficiency with progression to death, or permanent ventilation before age 2.8 In preparation for human trials, a major step was demonstrating that AAV9 reached the nerve cells of the brainstem and spinal cord.
It was confirmed in SMA murine models that systemically delivered self-complementary AAV9 (scAAV9) crossed the blood-brain barrier and achieved high levels of neuronal transduction.9,10 Preclinical studies, delivering scAAV9-SMN to SMA pups, demonstrated positive effects on survival, growth, and neuromuscular transmission.11,12 Two key observations from these studies worth noting were both time of intervention and dose-response effects on survival. Early treatment at postnatal day 2 extended the lifespan from 15 days to >250 days.11
The investigational new drug (IND) provided for an open-label, doseascending clinical gene-therapy trial starting May 13, 2014. A dose-ranging study of scAAV9.chicken b-actin (CB).SMN (START trial) at low (n = 3; 6.7 1013 mg/kg) and high (n = 6; 3.3 1014 mg/kg) dose was approved.13 Enrollment included symptomatic SMA1 infants with 2 SMN2 copies and onset of symptoms before 6 months of age in the absence of permanent ventilation. At the trial start, prednisolone was not included in the protocol.
On day 9 after SMN gene delivery to the first patient, serum chemistries showed the alanine aminotransferase (ALT) increased 16 > normal. Based on these findings, protocol amendments were submitted to the FDA and included prednisone, 1 mg/ kg per day begun 24 h before gene delivery and as a safety measure, reduced viral load for high dose from 3.3 1014 vg/kg to 2.0 1014 vg/kg.
This is now considered a "therapeutic dose" and adopted for other gene therapy clinical trials. The SMA gene therapy trial results perhaps exceeded expectations for the 15 SMA subjects enrolled (low dose n = 3; high dose n = 12).
At the study conclusion (December 2017), all infants were alive, and all 12 patients treated with the therapeutic dose were free of permanent ventilation.14 Treatment benefit was rapid: Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOPINTEND) increased 9.8 points at 1 month and 15.4 points at 3 months.
In the therapeutic dose, 11 sat unassisted, 9 rolled over, 11 fed orally and could speak, and 2 woke independently.13 Figure 1 shows mean CHOP-INTEND scores for 24 months for SMA patients treated at the therapeutic dose compared to natural history. These motor milestones are unprecedented in SMA1 infants and were a clear indication of robust widespread transduction of the motor neurons.
A key clinical observation that distinguished patients treated with therapeutic doses was the preservation and recovery of respiratory and oral motor skills. Efficacy correlated with early treatment and high CHOP-INTEND.15
The single patient treated at 7.9 months did not respond to therapy. Elevated serum aminotransferases were seen in 4 patients and were attenuated by corticosteroids.
In April 2018, Novartis entered an agreement to acquire AveXis and continued following the 15 subjects enrolled in the study. In May 2019, onasemnogene abeparvovec (Zolgensma) received FDA approval as the first-ever systemically delivered AAV gene therapy. Following approval, noteworthy events include the continued long-term monitoring of subjects enrolled in the START trial.
In the last data cut on December 31, 2019, 11 of 12 patients treated in the first trial at the therapeutic dose survived without the need for permanent ventilation. New milestones were documented, and the CHOP-INTEND score increased by 24.5 points.
Two additional patients gained the milestone of standing with assistance. At data cut, the oldest patient receiving the therapeutic dose was 5.6 years of age, now 5.2 years since gene transfer.
To further assess the impact of early intervention, a new multicenter study of presymptomatic infants (%6 weeks of age) with 2 or 3 copies of SMN2 has begun (SPR1NT).
Treatment at a mean age of 20 days shows improvement in CHOP-INTEND scores >50 in all subjects with survival up to 18 months (Figure 2). In 2019, SMA was added to the Recommended Uniform Screening Panel (RUSP), and numerous states have adopted this policymaking gene therapy available to newborn infants within the first few weeks after birth.
In Ohio, 5 presymptomatic patients have been treated with onasemnogene abeparvovec, with the oldest now 12 months of age. The future looks bright for SMA patients. The success of SMA type 1 gene therapy was extended to SMA patients R6 months to <5 years with three copies of SMN2, and eligibility required the ability to sit independently in the absence of walking without support.
Three doses of scAAV.CB.SMN was to be administered intrathecally. Low- and mid-dose cohorts completed enrollment, but the high dose was put on partial hold by the FDA.
This was in response to an AveXis-reported preclinical study in nonhuman primates (NHPs) receiving intrathecal vectors. They found dorsal root ganglia (DRG) mononuclear cell inflammation, sometimes accompanied by neuronal cell body degeneration or loss. The hold did not affect intravenous Zolgensma clinical trials. Recent studies from the Wilson lab18 provide some further insight.
Upon AAV vector infusion into the subarachnoid space in NHPs, pathology in the DRG is a consistent finding in virtually all animals. Similar findings have been seen, even in some studies when higher doses are administered systemically. However, in these NHPs, there is a notable absence of any clinical sequelae while using therapeutic transgenes.
Thus, in the context of risk-benefit, the response to gene delivery in clinical SMA disease13 outweighs concerns about the inflammatory infiltrate seen in DRG. DMD DMD is an X-linked, degenerative muscular dystrophy caused by mutations in the DMD gene.19
The gene product, dystrophin, is an integral cytoskeletal protein, anchoring the contractile actin filaments to the sarcolemma of both skeletal and smooth muscle cells.
Throughout the disease, loss of dystrophin results in membrane fragility, cycles of necrosis and regeneration, diminished regenerative capacity, and muscle replacement by fibrosis.20 Clinically, progressive muscle weakness results in loss of ambulation at 914 years of age and cardio-respiratory insufficiency leading to death in the second or third decade.21
Elevation of muscle creatine kinase (CK) indicative of ongoing dystrophic pathology is evident at birth.22 CK declines in nonambulatory patients as skeletal muscle is replaced by fibrosis.
The large size of the DMD gene renders it susceptible to spontaneous mutations. The incidence is 1:5,000 male births.22,23 Functional dystrophin production of a varying amount results from targeting specific mutations amenable to exon-skipping.24 Although some antisense oligonucleotide (AON) therapies have FDA approval,25 the applicable patient population and preservation of function remain limited. Gene replacement therapy is potentially an improved strategy for targeting a broader cohort of DMD patients.
A major hurdle has been packaging the large DMD gene in AAV vectors limited to <5 kb. To circumvent this challenge, designs for miniaturizing the dystrophin gene have been developed.
The adoption of a truncated version of dystrophin as a possible treatment for DMD is based on a Becker muscular dystrophy patient who remained ambulatory for seven decades despite a deletion of nearly half his DMD gene.26
Currently, transgenes incorporating diverse numbers of spectrin repeats (SRs) and hinges and delivery using different AAV serotypes are undergoing assessment of safety and efficacy in simultaneous systemic gene therapy trials (Table 1) sponsored by Sarepta Therapeutics, Pfizer, and Solid Biosciences, summarized below. Sarepta Therapeutics has recently reported the results of its phase 1/2 open-label, safety, and tolerability trial, conducted at Nationwide Children's Hospital (ClinicalTrials.gov: NCT03375164).27 Enrollment included 4 DMD boys, mean age 4.8 years, with mutations between exons 18 and 58, taking prednisolone for R12 weeks, who received rAAVrh74.
MHCK7.micro-dystrophin (SRP-9001) delivered through an extremity vein. Subjects with AAVrh74 total binding antibody titers >1:400 were excluded. The protocol included a single dose of daily prednisone, 1 mg/kg, starting 1 day before gene delivery, and continuing for 30 days (or more if needed). A single dose of SRP9001, 2.0 1014 mg/kg was infused. The AAVrh74 serotype in combination with the tissue-specific MHCK7 promoter with an a-myosin enhancer predicted high levels of expression in both skeletal and cardiac muscle.
The micro-dystrophin transgene contains SRs 13 that bind to the sarcolemma leading to improvement in sarcolemmal binding and force production, as well as SR 24 and hinges 1, 2, and 4.28At 12 weeks, a gastrocnemius muscle biopsy with comparison to baseline showed a mean micro-dystrophin expression of 81.2% of muscle fibers, with mean intensity of 96%.
Western blot (WB) showed a mean expression of 74.3% without adjustment for fat or fibrosis and 95.8% with adjustment. Serum CK remained decreased in all subjects (range 46%85%) with functional improvement by a mean of 5.5 points in the North Star Ambulatory Assessment (NSAA) at 1 year.
There was also improvement in time to climb 4 stairs and run/walk 100. Histological findings revealed absent central nucleation and ring myofibers and a reduction in the percentage of collagen content in muscle post-treatment compared with baseline (mean 26.7% ± 8.4%).
The adverse event (AE) profile was minimal. Three patients had transient elevation of g-glutamyltransferase that resolved with corticosteroids. A total of 18 events were considered treatment-related; the most common was vomiting (50%).
The cause for vomiting is unclear but did not correlate with AAV immunity. The safety and efficacy profile encouraged long-term follow-up and an ongoing phase 2, randomized, placebo-controlled, 96-week extension study with a larger sample size (ClinicalTrials.gov: NCT03769116).
Pfizer pharmaceutical company provided results of its ongoing safety and tolerability trial of PF-06939926 at the recent American Society of Gene and Cell Therapy (ASGCT) meeting, on May 15, 2020. rAAV9.mini-dystrophin was delivered to 9 DMD boys, ages 6.2 to 12.8; all were taking daily glucocorticoids, without pre-existing neutralizing AAV9 antibodies.
The dose-ascending schedule included 1.0 1014 mg/kg (n = 3) or 3.0 1014 mg/kg (n = 6). The muscle biopsies at baseline compared mini-dystrophin to normal using liquid chromatography-mass spectrometry (LCMS): 2 months 20% and at 12 months 24% (n = 3).
At high dose, expression was 35% (n = 6) and 52% (n = 3), in months 2 and 12, respectively. Immunofluorescence (IF) results were in the same range for samples tested at high and low doses with a slight decrease at 12 months. The NSAA for 3 subjects at 1 year showed a median increase of 3.5 points from baseline.
The fat fraction estimated by MRI at 12 months showed a reduction of 8% at a high dose (n = 3) and was unchanged at a low dose. The AE profile was more complex.
More than 40% of participants experienced vomiting, nausea, decreased appetite, and pyrexia. Because of the report of acute kidney injury involving atypical hemolytic uremic syndrome (aHUS)-like with complement activation requiring hemodialysis and eculizumab, the trial was originally put on hold by Pfizer to enable protocol amendments (June 2019).
In another subject, thrombocytopenia with aHUS-like complement activation required platelet transfusion and eculizumab. Pfizer has announced a planned phase 3, randomized, multicenter, double-blind, placebo-controlled trial inclusive of 99 subjects (C3391003) registered in ClinicalTrials.gov: NCT04281485.
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