Current Clinical Applications Of In Vivo Gene Therapy With AAVs Part 3
Jul 24, 2024
GAN
GAN is an autosomal recessive neurodegenerative disorder of the central and peripheral nervous system that typically presents with progressive weakness and ataxia. Individuals also suffer sensory loss and loss of ambulation and succumb to respiratory failure.62
Recessive inheritance and memory are two topics that seem to have no direct connection, but there is a close relationship between them. Recessive inheritance refers to the genetic inheritance phenomenon that exists in the genome but does not cause obvious phenotypic changes. Memory refers to the ability of humans to acquire, store, and retrieve information during cognitive processes. So, how are these two topics connected?
First of all, recessive inheritance and memory are both products of the nervous system. There are tens of billions of neurons in our brain, which transmit information through the preganglionic substantia nigra and synapses. The connection between neurons and the transmission efficiency of synapses are all affected by genes. Therefore, we can say that the development and function of the nervous system is a bridge between recessive inheritance and memory.
Secondly, recessive inheritance and memory also have a mutual influence relationship. Some studies have shown that gene variation can affect the performance of memory. For example, the loss or mutation of certain genes can affect the formation and stability of synapses in the brain, thereby affecting people's memory. At the same time, memory training can also affect recessive inheritance. Some experiments have shown that under certain circumstances, the plasticity of memory can affect the expression and mutation of specific genes, thereby changing the phenotypic characteristics of individual offspring.
Finally, both recessive inheritance and memory can be changed and improved. Although we cannot change our genome, we can improve our memory by creating a favorable environment and practice. For example, a healthy lifestyle, good sleep, and cognitive training can improve memory. Similarly, some new technologies and treatments, such as gene editing and gene therapy, can also be used to treat and improve recessive inheritance-related conditions.
In summary, there is a close relationship between recessive inheritance and memory. Although we cannot completely control our genetic inheritance, we can change and improve our memory by creating a favorable environment and practice, which is also something we should be positive about. It can be seen that we need to improve our memory, and Cistanche can significantly improve memory because Cistanche can also regulate the balance of neurotransmitters, such as increasing the levels of acetylcholine and growth factors, which are very important for memory and learning. In addition, Cistanche can also improve blood flow and promote oxygen delivery, which can ensure that the brain obtains adequate nutrition and energy, thereby improving brain vitality and endurance.
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Based on neuropathological studies, there is a loss of gigatons expression that affects the cerebellar cortex, brainstem, and posterior columns of the spinal cord, making it an ideal candidate disorder for intrathecal delivery of AAV9 gene transfer (reviewed by Bailey et al.63).
Gigaxonin is required to organize and degrade intermediate filaments and leads to enlarged axons with densely bundled intermediate filaments (reviewed by Bailey et al.63).
A phase 1 dose escalation study of intrathecal AAV9/GAN is underway at the National Institutes of Health (NIH; ClinicalTrials.gov: NCT02362438).
Future Outlook
Nearly 2 decades after the initial intracerebral gene-transfer trials using AAV2, methods for gene transfer to the CNS have greatly expanded. When focal gene transfer is desired, such as for PD and AADC, more accurate gene transfer to a greater volume of brain tissue can be achieved with AAV2 vectors using methods such as MRI-guided convection-enhanced delivery.64–66
However, most CNS disorders would ideally require broad and efficient gene transfer to the entire CNS. The discovery of newer AAV capsids, such as AAV9, has permitted a much wider degree of gene transfer than multiple stereotaxic injections.9,10,67–69 Aside from numerous studies in animal models, the application of AAV9 has been demonstrated in clinical trials after an intrathecal injection (as for GAN) or by an intravenous injection (as for SMA13).
It is anticipated that the use of AAV9 or similar AAV capsids will broaden the application of gene therapy to more CNS disorders shortly. However, whereas AAV9 has greatly expanded the ability to treat a larger number of CNS disorders, it still targets a minority of cells throughout the brain.70
Looking forward, a newer generation of AAV capsids with greater CNS targeting efficiency would increase the effectiveness of CNS-directed gene therapy treatments, as well as expand the number of diseases that could potentially be treated with gene therapy.
Clinical In Vivo Gene Therapy for Ocular Disorders
Gene therapy gained its place in mainstream medical practice following FDA approval of Luxturna, an AAV2-based treatment for the inherited retinal disease (IRD) retinal pigment epithelium (RPE)65-LCA (LCA2).
This success was based on decades of work by multiple groups, one of which went on to commercialize the product.71–76 Treated patients exhibited life-changing improvements in light sensitivity and visually guided behavior.
Detailed summaries of RPE65 biology, preclinical studies in animal models, and the treatment of LCA2 with gene therapy by multiple groups are already published.77,78
The dramatic success of this program catalyzed academia and industry alike to establish proof of concept that gene therapy could restore or preserve vision in animal models of other retinal diseases, including, but not limited to, AMD, choroideremia, ACHM, retinitis pigmentosa, and XLRS.79–85
Interestingly, however, these preclinical successes have not consistently translated to clinical outcomes as robust as those observed in LCA2 patients.86–92 Whereas the reasons for this discrepancy have yet to be fully elucidated, insufficient transgene expression mediated by AAV in the target cells and/or immune response likely played a role.
Future success in the retinal gene therapy space and the broader gene therapy field will depend on identifying feasible therapeutic dose ranges that are based on the proven ability to (1) target the appropriate cell type in a primate retina and (2) drive sufficient levels of therapeutic transgene expression. Whereas many factors contribute to AAV's tropism, transduction efficiency, and associated immune response in the retina, the route of delivery is especially critical. A summary of current and developing approaches, their advantages and disadvantages, and relevant clinical examples are discussed below.
Subretinal Injection (SRI)
SRI is employed in the majority of clinical trials because it allows for placement of the therapeutic in situ (in a surgically created space between photoreceptors (PRs) and RPE referred to as a subretinal "bleb").
The majority of IRDs are caused by mutations in PR-specific genes. In addition to its proximity to the most common clinical target cells (i.e., RPE and PR), SRI is attractive because of this compartment's immune privilege. Unlike systemically delivered AAV, subretinally delivered vectors elicit a relatively reduced immune response akin to anterior chamber-associated immune deviation (ACAID).93,94
However, SRI is a challenging technique, described as "almost a subspecialty unto itself." 95 It requires a vitrectomy (removal of vitreous humor) and retinotomy (passage of the needle through the retina), which can be associated with complications, such as retinal tears, cataract progression, or retinal/choroidal hemmorage.96
Creation of the subretinal bleb requires detaching the retina from the underlying RPE. The cone-exclusive fovea is especially sensitive to detachment. SRI of vector under the fovea of some LCA2 patients led to central retinal thinning and loss of visual acuity.75
Similar decreases in retinal thickness were observed in choroideremia patients.96 The surgical technique of subretinal gene therapy is predicated upon established subretinal procedures, such as subretinal tissue plasminogen activator (tPA) injection for subretinal hemorrhage associated with neovascular AMD.96
However, specific considerations must be made to adapt the technique to the particular characteristics of the retinal structure present in IRDs. Vitreoretinal surgeons well versed in subretinal gene therapy have reported the utility of intraoperative optical coherence tomography (OCT), allowing in vivo real-time feedback during surgical cases.97
The creation of the subretinal bleb with a microneedle (typically a 38- to 41-gauge, Teflon-tipped, either extendable or non-extendable, a cannula that is placed through a pars plana trocar) is a challenging and critical step in the procedure.98
The needle-penetration step has a narrow margin of error: excessively deep penetration of the needle tip can result in hemorrhage, cannula tip obstruction, unintentional suprachoroidal delivery of vector, or permanent RPE injury; however, too shallow needle penetration can create retinoschisis by intraretinal hydration during bleb formation.97
Some surgeons create a "pre-bleb" made with balanced salt solution (BSS) before the injection of the vector into this space, which may prevent the loss of the vector into the vitreous cavity during the bleb creation.
Another surgical consideration is the inherent difficulty of uniform and accurate volume delivery associated with the current transversal subretinal delivery method.
Vector volume can be affected by the use of a BSS pre-bleb, loss of vector from vitreous egress from the retinotomy site, and incomplete target vector volume delivery due to surgeon discretion (concern for foveal stretching or macular hole formation or other safety considerations).
In clinical trials where dose-escalation decisions are being made with small numbers of subjects, it may be challenging to appropriately make safety or efficacy decisions unless confirmation of precise and uniform vector volume delivery can be achieved in all patients dosed.
It is also important to note that, despite its relative immune privilege, AAV vectors are still capable of reaching an adverse effect level and eliciting host-cell responses in the subretinal space.
In phase I/II clinical trials for RPE65-LCA at University College London (ClinicalTrials.gov: NCT00643747), ocular inflammation was noted following SRI of 1 1012 vg of AAV2-RPE65.71,99
There was no inflammation noted in the University of Pennsylvania/University of Florida trial (ClinicalTrials.gov: NCT00481546). In the Nantes University Hospital trial (ClinicalTrials.gov: NCT01496040), which notably used a different AAV capsid (AAV4), inflammation was noted at 4.8 1010 vg.100 In Spark Therapeutics' phase III trial (ClinicalTrials.gov: NCT00999609), mild inflammation was observed at 1.5 1011 vg.76,101 Significant inflammation was also observed at 1 1011 vg in clinical trials for choroideremia.90,102
The different doses at which inflammation has been observed clinically may be attributed to differences in vector production and characterization, AAV capsid, and underlying retinal disease state.
As the focus has now shifted to evaluating gene therapies for IRDs where the target cells are PRs, it is worth considering the relationship between the abundance of the gene-replacement product and vector dosing.
For any gene therapy to be successful, sufficient therapeutic transgene expression (e.g., protein) levels must be achieved at doses that do not cause unmanageable inflammation. Ideally, a sufficient range between the minimum effective dose in animal models and the NOAEL (no observable adverse effect level) should exist such that a phase I/II dose-escalation study can be performed.
Logic dictates that IRDs caused by defects in retinal proteins expressed at relatively low levels may be more easily addressed by gene therapy.
A comparison of preclinical findings in large animal models of IRD versus clinical outcomes supports this concept, although published clinical outcomes remain scant to date. Preclinical studies sponsored by AGTC evaluated AAVRPGR (retinitis pigmentosa GTPase regulator) in the diseased canine model of RPGR X-linked retinitis pigmentosa (XLRP).103 Efficacy (improvements in fundus autofluorescence) was reported at doses as low as 1.8 109 vg,103, and inflammation was not observed until a dose of 4.5 1011 vg.103 Biogen's 6-month AAV-RPGR phase I/ II clinical trial results were presented at this year's ASGCT meeting.
It reported improvements in the visual fields in six treated XLRP patients, "exceptional visual improvement," and "evidence of possible outer segment regeneration" in one patient treated at the high dose (5 1011 vg). Manageable inflammation was only observed in this high-dose group.
Whereas not yet presented/published, AGTC's press release104 (date accessed 6-12-20) states that 9 out of 17 treated patients experienced improvements in vision, as measured by microperimetry and/or best-corrected visual acuity (BCVA) at 3 months postinjection. Only patients with submacular injections showed this improvement.
At 6 months postinjection, improvements in those nine patients were stable. In four of these patients (4/8 tested), there were improvements in visual sensitivity (i.e., microperimetry).
Proteomic analysis reveals there are approximately 2,000 molecules of RPGR per PR sensory cilium (PSC), making it the 1,087th most enriched protein in rods.105 In contrast, preclinical studies evaluating AAV-cyclic nucleotide-gated channel subunit beta 3 (CNGB3) in a dog model of CNGB3 ACHM revealed only modest improvements in retinal function at 5 1010 vg and inflammation at 5 1011 vg.
Phase I/II trials for CNGB3 ACHM were initiated in 2016 by both AGTC and MeiraGTx, but clinical outcomes have not yet been presented/published. Cyclic nucleotide-gated channels are highly expressed (61,000 molecules per PSC), making them much more abundant than RPGR.105
Preclinical studies in a sheep model of cyclic nucleotide-gated channel subunit alpha 3 (CNGA3) ACHM revealed robust improvements in retinal function at 1.8 1011 vg (lowest dose tested) and inflammation at 6.0 1011 vg.
Phase I/II clinical trials conducted at the University Hospital Tuebingen and Ludwig Maximilian University of Munich (ClinicalTrials.gov: NCT02610582) treated patients at the following doses: 1 1010 vg, 5 1010 vg, or 1 1011 vg.91
No unmanageable inflammation was reported, and modest improvements in visual acuity, contrast sensitivity, and chromatic discrimination thresholds were observed at all doses, consistent with biological activity mediated by vector.92
Doses required to confer therapy in preclinical studies were higher in CNGA3-ACHM relative to RPGR-XLRP, although the CNGA3-ACHM clinical results suggest that IRDs caused by defects in highly expressed genes may still be successful.
Some IRDs, such as MYO7A-associated Usher syndrome and ABCA4-associated Stargardt disease, are caused by mutations in genes for which coding sequences are too large to fit within a standard AAV vector (packaging capacity 5 kb).
Lentivirus, which can accommodate a larger payload (9.7 kb), was therefore chosen to address these IRDs in phase I/II clinical trials that began in 2011/ 2012. No reports of biological activity have been published to date for either trial.
This is thought to be attributed to the fact that lentivirus poorly transduces postmitotic PRs,106,107 the target cell in both USH1B and Stargardt. Efforts are currently underway by multiple groups to develop dual AAV vector platforms that will promote the delivery of large genes to PRs.108,109
Beyond monogenic disease, AAV is also being used to vectorize antivascular endothelial growth factor (VEGF) reagents to the retina via SRI to serve as a one-time treatment for the neovascular form of AMD (wet AMD).
This represents a potential improvement over the standard-of-care monthly intravitreal injections (IVIs) of VEGF inhibitors that can suffer from low compliance. REGENXBIO recently completed a phase I/IIa trial in which patients across five cohorts received doses of AAV8-anti VEGF fab between 3 109 vg and 2.4 1011 vg per eye. Dose-dependent increases in protein expression levels were observed at 1 month p.i.
There were no drug-related AEs and no clinical signs of an immune response or drug-related ocular inflammation. Whereas no clear signs of improvements were noted in cohort 1 (3 109 vg/eye) or cohort 2 (1 1010 vg/eye), patients in cohort 3 (6 1010 vg/eye) showed improvements in mean BCVA and central retinal thickness. 50% of patients in cohort 3 remain injection-free (i.e., no IVIs VEGF inhibitors) for up to 2 years post-treatment.
Most recently, results from cohort 5 (2.5 1011 vg/ eye) showed that 73% of patients remained injection-free for at least 9 months post-treatment. REGENXBIO has plans to modify its delivery approach.
Its goal is to deliver AAV to the subretinal space via a microcannula that accesses the retina posteriorly from the suprachoroidal space (see the section below) and in so doing, increase accessibility to the treatment (will not require vitrectomy/full-blown surgery). Another topic of interest within the IRD gene therapy field is the evolving analysis of risk to benefit for the subretinal approach.
An example is whether detachment of the cone-exclusive fovea via SRI is advisable. In certain IRDs characterized by the presence of severe functional deficits despite retinal preservation (e.g., GUCY2DLCA1), SRI of the macula poses an attractive risk-benefit ratio.110,111 Put simply, patients with completely dysfunctional foveal cones have less risk in terms of function and a large potential for gain as their preserved retinal laminar architecture makes them (1) more likely to tolerate surgical foveal detachment and (2) potentially more receptive to therapy.
This is in contrast with patients who retain cone function and have actively degenerating retina (i.e., retinitis pigmentosa). In the latter scenario, SRI of AAVs capable of laterally spreading beyond the margin of detachment may mediate therapeutic levels of gene expression in foveal cones while avoiding the risks associated with foveal detachment.
Indeed, extra-foveal SRI of such vectors (AAV44.9 based) promoted 98% transduction of foveal cones in macaque without the need to detach the fovea during surgery.112
In addition, these novel vectors that spread laterally from the injection bleb may also enable functional improvements across a greater expanse of the retina.112 Despite the surgical complexity, the many advantages that SRI offers (robust gene expression in outer retinal cells, delivery to a relatively immune-privileged site, its proven success in addressing IRDs, and novel capsid technologies being explored to further increase its safety) suggest that this delivery approach will continue to be used for some time.
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