Juggling Safety and Efficacy: Finding Ways to Achieve Both

Abstract

Direct intraparenchymal injection of recombinant adeno-associated virus (rAAV) into the brain is a straightforward way to bypass the blood–brain barrier (BBB), delivering gene therapeutics to the brain. This route of administration (ROA) of rAAV has been utilized in clinical trials for diseases such as aromatic L-amino acid decarboxylase deficiency, Parkinson's disease, Batten disease (CLN2 mutations), and metachromatic leukodystrophy (MLD).¹ In general, a single injection results in relatively localized gene delivery that can suffice to target a specific afflicted brain substructure, and injections at multiple sites can cover a broader region of the central nervous system (CNS). Compared with targeting the CNS by intravascular delivery of certain BBB-crossing AAV capsids, intraparenchymal injection is far less susceptible to circulating anti-AAV neutralizing antibodies (NAbs), requires much less vector, and largely avoids certain toxicities related to high-dose systemic delivery.

In this issue of Human Gene Therapy, Rosenberg et al. ² reported a formal dose-escalating safety/toxicology study on intraparenchymal delivery of rAAVrh.10-hARSA, an AAVrh.10-based vector expressing human arylsulfatase A (ARSA) for treating MLD, to nonhuman primates (NHPs). This study builds upon previous studies showing that the therapeutic regimen is efficacious in an MLD mouse model, and results in widespread expression of ARSA in the CNS of NHPs.^3,4 Although no significant adverse effects were observed in animals receiving phosphate-buffered saline or low dose (2.85 × 10¹⁰ genome copies [gc] total; 2.4 × 10⁹ gc per site × 12 sites) of rAAVrh.10-hARSA, CNS abnormalities were noted in the animals receiving high dose (1.5 × 10¹² gc total; 1.3 × 10¹¹ gc per site × 12 sites) of rAAVrh.10-hARSA or control rAAVrh.10 not expressing ARSA (rAAVrh.10Null). These include localized lesions revealed by magnetic resonance imaging (MRI), which were associated with infiltrating T cells, B cells, and macrophages at the sites of catheter infusions. However, there was no adverse clinical consequence observed during the study period of 1 year, and other measurements appeared normal, including general health conditions, behavior, clinical serum chemistry, and hematology assessments.

In an earlier safety study in NHPs, Zerah et al. ⁵ reported that direct brain injections of rAAVrh.10-hARSA at a similar high dose (1.1 × 10¹² gc total; 9.2 × 10¹⁰ gc per site × 12 sites), but not at a low dose (2.2 × 10¹¹ gc total; 1.8 × 10¹⁰ gc per site × 12 sites), also resulted in brain abnormalities detected by MRI at the injection sites 3 months postinjection.⁵ This study informed a phase I/II clinical trial for MLD, that is, the low dose (2.2 × 10¹¹ gc total) with a good safety profile was scaled 20-fold by brain weight to derive two doses used in the clinical trial: 4 × 10¹² gc total (3.3 × 10¹¹ gc per site × 12 sites) and 1 × 10¹² gc total (8.3 × 10¹⁰ gc per site × 12 sites). Despite significant and durable restoration of ARSA activity in the cerebrospinal fluid (CSF), positive clinical impact was not observed.⁶ Therefore, this study by Rosenberg et al. along with previous preclinical and clinical work suggested a dose limit of safe intraparenchymal injection of rAAVrh.10 to the white matter of primate CNS, which may be a bottleneck to achieve efficacious therapeutic outcome for some diseases such as MLD.

Because rAAVrh.10Null with a nontranslatable vector genome of the same high dose also resulted in CNS abnormalities, the capsid, rather than the transgene, was likely the culprit. Therefore, novel capsids that can confer more potent gene delivery after intraparenchymal injection would enable efficacious gene delivery at safe doses. It is conceivable that such “super transducers” are achievable through capsid development effort encompassing diverse approaches. For example, Hsu et al. ⁷ recently reported a neurotropic capsid named AAVv66, an AAV2 variant identified in human tissue samples by high-throughout long-read DNA sequencing. After unilateral intrahippocampal injection in mice, AAVv66 vector showed >10-fold transduction in hippocampus than an AAV2 counterpart.⁷ In addition to discovering natural AAV capsids, other capsid engineering approaches such as directed evolution also hold great promise for developing capsids that allow for efficacious gene delivery with lower doses and thus a better safety profile. Nevertheless, potential toxicities relating to transgene expression, even in the immune-privileged CNS space, are possible and should be carefully evaluated, as exemplified in a recent report showing gain of toxicity by long-term AAV9-mediated SMN overexpression in the mouse motor neurons.⁸

To target the broad CNS, alternative ROAs other than or in combination with intraparenchymal injection may be advantageous. rAAV delivery to the CSF has proven to be a safe and effective means to achieve widespread CNS transgene expression in sheep and NHPs.^9,10 As demonstrated by Rosenberg et al. and other previous studies, neither intraparenchymal nor CSF delivery of rAAV completely restricts biodistribution exclusively within the CSF space; “leakage” to the peripheral appears inevitable, which will elicit circulating NAbs.^2,11 Interestingly, vector-induced NAbs in CSF are usually not detected,¹¹ suggesting the possibility of redosing.

Taking advantage of the highly elaborate architecture of brain microvasculature, intravascular delivery of certain BBB-crossing AAV capsids can achieve widespread brain gene delivery. However, this ROA is prone to circulating NAbs. In addition, it requires a high dose proportional to body weight, which poses a significant manufacturing burden. High dose of systemic injection of rAAV was also associated with severe adverse effects in several recent animal studies and clinical trials.^12
–14 Developing potent BBB-crossing AAV capsids to enable CNS gene delivery after systemic injection of lower doses is a mainstream research effort in the gene therapy field. Although some capable capsids prove to suffer from species-specific limitations that are largely attributable to the capsid screening platform in model animals,^15
–17 developing such potent BBB-crossing AAV capsids suitable for human use is feasible. Besides capsid development, refined transgene cassette design can also help to minimize off-target transduction,¹⁸ further improving the safety profile of CNS-targeting systemic delivery.

AAV gene therapy for each disease has a unique set of parameters to consider, including disease foci, therapeutic transgene expression threshold, and potential toxicities when the transgene is expressed in off-target tissue/cell types, among others. Although a generalizable treatment regimen is unlikely to prevail, common themes regarding safety and efficacy after rAAV administration, especially gained from large animal studies and clinical trials, will continue to emerge and inform on the future refinement of human gene therapy.

References

Deverman

, Ravina

, Bankiewicz

, et al. Gene therapy for neurological disorders: progress and prospects. Nat Rev Drug Discov, 2018; 17:641–659.

Rosenberg

, Chen

, De

, et al. Safety of direct intraparenchymal AAVrh.10-mediated central nervous system gene therapy for metachromatic leukodystrophy. Hum Gene Ther, 2021; 32:563–580.

Piguet

, Sondhi

, Piraud

, et al. Correction of brain oligodendrocytes by AAVrh.10 intracerebral gene therapy in metachromatic leukodystrophy mice. Hum Gene Ther, 2012; 23:903–914.

Rosenberg

, Sondhi

, Rubin

, et al. Comparative efficacy and safety of multiple routes of direct CNS administration of adeno-associated virus gene transfer vector serotype rh.10 expressing the human arylsulfatase A cDNA to nonhuman primates. Hum Gene Ther Clin Dev, 2014; 25:164–177.

Zerah

, Piguet

, Colle

, et al. Intracerebral gene therapy using AAVrh.10-hARSA recombinant vector to treat patients with early-onset forms of metachromatic leukodystrophy: preclinical feasibility and safety assessments in nonhuman primates. Hum Gene Ther Clin Dev, 2015; 26:113–124.

Sevin

, Thomas

, Nathalie

, et al. Intracerebral gene therapy in children with metachromatic leukodystrophy: results of a phase I/II trial. Mol Genet Metab, 2018; 123:S129.

Hsu

, Brown

, Loveland

, et al. Structural characterization of a novel human adeno-associated virus capsid with neurotropic properties. Nat Commun, 2020; 11:3279.

Van Alstyne

, Tattoli

, Delestrée

, et al. Gain of toxic function by long-term AAV9-mediated SMN overexpression in the sensorimotor circuit. Nat Neurosci 2021.

Taghian

, Marosfoi

, Puri

, et al. A safe and reliable technique for CNS delivery of AAV vectors in the cisterna magna. Mol Ther, 2020; 28:411–421.

10.

Rosenberg

, Kaplitt

, De

, et al. AAVrh.10-mediated APOE2 central nervous system gene therapy for APOE4-associated Alzheimer's disease. Hum Gene Ther Clin Dev, 2018; 29:24–47.

11.

Bey

, Deniaud

, Dubreil

, et al. Intra-CSF AAV9 and AAVrh10 administration in nonhuman primates: promising routes and vectors for which neurological diseases?. Mol Ther Methods Clin Dev, 2020; 17:771–784.

12.

Hordeaux

, Buza

, Dyer

, et al. Adeno-associated virus-induced dorsal root ganglion pathology. Hum Gene Ther, 2020; 31:808–818.

13.

Hinderer

, Katz

, Buza

, et al. Severe toxicity in nonhuman primates and piglets following high-dose intravenous administration of an adeno-associated virus vector expressing human SMN. Hum Gene Ther, 2018; 29:285–298.

14.

Wilson

, Flotte

. Moving forward after two deaths in a gene therapy trial of myotubular myopathy. Hum Gene Ther, 2020; 31:695–696.

15.

Hordeaux

, Wang

, Katz

, et al. The neurotropic properties of AAV-PHP.B are limited to C57BL/6J mice. Mol Ther, 2018; 26:664–668.

16.

Huang

, Chan

, Tobey

, et al. Delivering genes across the blood-brain barrier: LY6A, a novel cellular receptor for AAV-PHP.B capsids. PLoS One, 2019; 14:e0225206.

17.

Batista

, King

, Reardon

, et al. Ly6a differential expression in blood-brain barrier is responsible for strain specific central nervous system transduction profile of AAV-PHP.B. Hum Gene Ther, 2020; 31:90–102.

18.

Xie

, Xie

, Zhang

, et al. MicroRNA-regulated, systemically delivered rAAV9: a step closer to CNS-restricted transgene expression. Mol Ther, 2011; 19:526–535.