The Challenge of Gene Therapy for Neurological Diseases: Strategies and Tools to Achieve Efficient Delivery to the Central Nervous System

Abstract

For more than 10 years, gene therapy for neurological diseases has experienced intensive research growth and more recently therapeutic interventions for multiple indications. Beneficial results in several phase 1/2 clinical studies, together with improved vector technology have advanced gene therapy for the central nervous system (CNS) in a new era of development. Although most initial strategies have focused on orphan genetic diseases, such as lysosomal storage diseases, more complex and widespread conditions like Alzheimer's disease, Parkinson's disease, epilepsy, or chronic pain are increasingly targeted for gene therapy. Increasing numbers of applications and patients to be treated will require improvement and simplification of gene therapy protocols to make them accessible to the largest number of affected people. Although vectors and manufacturing are a major field of academic research and industrial development, there is a growing need to improve, standardize, and simplify delivery methods. Delivery is the major issue for CNS therapies in general, and particularly for gene therapy. The blood–brain barrier restricts the passage of vectors; strategies to bypass this obstacle are a central focus of research. In this study, we present the different ways that can be used to deliver gene therapy products to the CNS. We focus on results obtained in large animals that have allowed the transfer of protocols to human patients and have resulted in the generation of clinical data. We discuss the different routes of administration, their advantages, and their limitations. We describe techniques, equipment, and protocols and how they should be selected for safe delivery and improved efficiency for the next generation of gene therapy trials for CNS diseases.

Introduction

The central nervous system (CNS) is protected by a unique microvasculature, the blood–brain barrier (BBB), composed of endothelial cells connected by tight junctions and adherent processes. The BBB controls brain homeostasis and ion and molecule movements thus protecting the CNS against potential intruders. The restrictive nature of the BBB provides an obstacle for drug delivery to the CNS, and major efforts have been made to generate methods to modulate or bypass the BBB for delivery of therapeutics. Contrarily, some pathologies of the CNS including stroke, multiple sclerosis, brain traumas and neurodegenerative disorders, alter the BBB, causing it to become more permeable, allowing the entry of molecules that can induce inflammatory responses and lead to neuronal damage.^1,2

Gene therapy has been applied to several CNS diseases, including neurodegenerative³ and neurodevelopmental disorders,^4,5 and also for diverse conditions such as epilepsy,⁶ glioblastoma,⁷ and pain.⁸ Increasingly, gene therapy products can be tailored to counter the pathophysiological mechanisms of particular disease mechanism, including the use of gene replacement,^9

–13 gene silencing,¹⁴ transplicing,¹⁵ modulation of cellular pathways to improve phenotype,^16

–19 or expression of suicide gene.²⁰

Gene therapy products can be delivered by various routes of administration, using either ex vivo or in vivo strategies. Ex vivo gene therapy involves autologous transplantation of hematopoietic stem cells corrected by genetic modification of lentivirus (hematopoietic stem cell-gene therapy [HSC-GT]) outside the body and subsequent transplantation of the cells back into the patient. Efficacy for HSC-GT has been shown in clinical trials for leukodystrophies (adrenoleukodystrophy [ALD], metachromatic leukodystrophy [MLD]).^21,22 Therapeutic action involves either production of the therapeutic protein by donor-derived cells that can migrate into the CNS (permanent source of the missing enzyme), and/or modulation of the immune environment (replacement of microglial cells and/or perivascular macrophages). In vivo gene therapy requires direct introduction of the vector (carrying the therapeutic gene) into the patient (Fig. 1). Intravenous (IV) administration is a common route of administration for in vivo gene therapy but for CNS diseases the limited capacity of gene delivery systems to cross the BBB remains a significant obstacle. Strategies such as disruption of the BBB integrity (by osmotic or biochemical means) or improvement of viral vector capsids continue to be developed to enhance peripheral administration. Direct delivery into the parenchyma of the brain or the cerebrospinal fluid (CSF) bypasses the BBB and permits more targeted gene delivery. After intrathecal (IT) or intracerebroventricular (ICV) administration, the therapeutic vector enters the CSF and is delivered throughout the CNS (at least to tissue adjacent to CSF spaces). After intraparenchymal administration, the therapeutic vector enters the brain parenchyma and is delivered locally into brain cells. The diffusion of the therapeutic product is limited around the injection site; however, secretion uptake may improve diffusion, notably the case for most lysosomal storage disease enzymes.

Figure 1.

(a) Circulation of the CSF. Secretion by the choroid plexus and circulation within the ventricles to the foramen of Magendie and Lushka (black arrows) to the extraventricular subarachnoid spaces. Then circulation around the brain and the spinal cord (blue arrows), to the resorption spaces (mainly the superior sagittal sinus). (b) Intrathecal injection targeting the spinal cord. (c) Injection within the cisterna magna targeting the more superficial area of the brain (mainly the cortical zone). (d) Intraventricular injection targeting the deepest regions of the brain (mainly the basal ganglia and the subventricular nucleus). Notice that the hippocampus region can be theoretically reached by cisternal or intraventricular injection. (e) Following the CSF flow, a part of the vectors will reach the resorption areas and join the cerebral blood stream and then the cerebral circulation to the peripheral organs. CSF, cerebrospinal fluid.

The vector of choice for in vivo CNS delivery is the adeno-associated viral (AAV) vector. Recombinant AAVs have been widely used for CNS gene therapy, demonstrating safety, stable, and long-term expression and some degree of neuronal tropism relevant for many therapeutic applications. A large body of preclinical results have been obtained, particularly in large animals like dogs, cats, and nonhuman primates (NHPs) that have demonstrated feasibility for clinical use.²³

Wild-type AAVs are nonenveloped parvovirus, which are characterized by an icosahedral capsid and a 4.7 kb single-stranded DNA genome. To complete a replication cycle, AAVs require coinfection by a helper virus like adenovirus or herpes virus. AAVs infect humans and other species including the NHP.²⁴ Natural infection with AAV is not known to lead to disease, although there is controversy concerning hepatocellular carcinoma.^25,26 The minimal sequence needed to generate recombinant AAVs is restricted to the 145 bp within the inverted terminal repeats (ITRs) flanking the transgene.²⁷ The overall capacity of AAV to package an ITR-flanked genome productively is the approximate size of the wild-type AAV genome (i.e., 4.7 kb).

Overall AAV vectors have been used and have proven their safety and low immunogenicity tolerance in >200 human studies.²⁸ Efficiency of an AAV administration is determined mainly by the capsid that directs the tropism of the virion, but can be impacted by the route of administration.²⁹ It is also well established that the specificity of transgene expression is dependent on both the capsid and the regulatory elements present in the vector.³⁰ There are a large number of AAV serotypes based on capsid structure.³¹ AAV2, the first AAV serotype to be used as a replication-defective vector, has been the most extensively characterized. Other AAV serotypes developed later as vectors use a cross-packaging system, in which genomes flanked by AAV2 ITRs are packaged in other AAV capsids. These serotypes have a wide variety of tissue and cell tropism.³² For gene transfer to the CNS the most frequently used capsids have been AAV1,³³ AAV2,^34
–36 AAV5,³⁷ AAV9,¹² and AAVrh.10.⁹ AAV9 was shown to naturally cross the BBB but limited expression of therapeutic genes in the CNS after a single IV injection of vector.^38,39 Of importance, the first clinical report of gene-replacement therapy for spinal muscular atrophy (SMA) type I has demonstrated the safety and efficacy of this approach.¹² However, the efficacy of brain transduction efficacy may vary with the patient age at treatment. There are ongoing efforts to engineer capsids with improved capacity to cross the BBB. AAV variants^40,41 such as AAV.PHP.B and AAV-B1^12,42 have resulted in superior capacity for CNS targeting, at least in the animal models tested.⁴³

Lentiviral vectors (LV) are members of the Retroviridae family, and based either on human immunodeficiency virus (HIV), nonprimate primates (simian immunodeficiency virus), or others such as equine infectious anemia virus (EIAV).⁴⁴ The viral genome contains two long terminal repeats (LTR), with elements required for gene expression, reverse transcription, and integration into host chromosome.⁴⁵ Different pseudo types of LV with different envelopes allowing for different viral tropism have been developed. The most frequently used is the vesicular stomatitis virus glycoprotein (VSV-G), which has a broad tropism in vitro and neuronal and glial tropism in vivo. ^46,47 Lentiviruses are able to penetrate the intact nuclear membrane through nuclear pores, do not require cell division, and can efficiently infect quiescent cells.^48,49 This ability to transduce dividing and nondividing cells, long-term stable expression through transgene integration into the chromosomes of host cells, and their large cloning capacity make LVs desirable vectors for gene therapy.⁵⁰ They also have a cloning capacity of 9.7 kb.

LVs are particularly useful for ex vivo gene therapy applications. Hematopoietic stem cells (HSCs) can be stably transduced using LVs; allowing for stable, indefinitely persisting expression within the host cell, despite repeated cell division. This characteristic has been widely applied to CNS lysosomal storage diseases or ALD.^51,52 Random integration in the genome of host cells is associated with a potential genotoxicity risk, as previously observed with retroviruses.^53,54 However, long-term follow-up of gene therapy trials has not identified adverse events associated with insertional mutagenesis.^55
–57 In vivo use of lentivectors for CNS applications has been more limited. Because of their capacity to transduce neurons, they have been tested for the treatment of Alzheimer's disease (AD) and Parkinson's disease (PD).^58
–60 For this review, we focused more on AAV because they have been more widely used in CNS targeting for large animals and human clinical trials.

Evaluation of Administration Routes in Preclinical Studies

Translation to clinical application after proof-of-concept in mouse models of the disease most often requires efficacy and tolerance studies in large animals.

Many species have been used⁶¹ for preclinical development. Affinity for the different types of brain cells in large animal species remains a major issue. The choice of the large animal models to be used is mainly based on the use of a large animal model of the disease when available^62

–65 otherwise on anatomical aspects to mimic the best situation to be reproduced for future clinical trials.

When the gene therapy product is administered by injection in the blood stream or the CSF, any suitable species of large animal can be used. However, when delivery is based on direct intraparenchymal (IP) injection into the brain for characterizing biodistribution, toxicity, diffusion, and affinity with different types of brain cells, pigs, sheep, and NHPs are preferred species for modeling trajectories and efficient targeting. In all cases it must be emphasized that the use of large animals is strictly limited for ethical reasons and should be performed in good laboratory practice (GLP) or GLP-like conditions to be usable for regulatory documents.

Intraparenchymal delivery

The principle studies of IP gene delivery in large animals are given in Table 1. Direct IP injection—either direct perfusion or convection enhanced delivery (CED)—allows targeted delivery in CNS regions while bypassing the BBB. However, IP injections remain more complex to perform than ICV or IT techniques for several reasons.⁶⁶ The choice of the target region is always a challenging balance between efficacy and safety. Therefore, the specific anatomy and pathophysiology of the disease should be considered when choosing target sites. Targeting the white matter in multiple sites may enhance vector spreading.¹¹ In particular, AAV vectors are readily transported along axons that facilitate the distribution of the therapeutic gene.⁶⁷ The spreading and directionality of AAV transport are serotype dependent.⁶⁸ AAV2, which has been widely used in IP gene delivery, resulted in anterograde transport⁶⁸ of vector particles from basal ganglia to cortex in NHP.^69,70 In contrast, AAV6 is axonally transported exclusively in a retrograde direction,⁶⁸ whereas AAV9 shows a bidirectional transport and is dose dependent.⁷¹

Table 1.

Intraparenchymal delivery in large animal models

Transgene/Pathology	Target	Specie	Age	Number	Vector/Buffer	Dose	Volume	Speed	Results	Article
ARSA for MLD	Three areas of the centrum semiovale white matter or in the deep gray nuclei (caudate nucleus, putamen, thalamus)	NHP	2- to 3-years old	6	AAV2–5	1.9.10¹² vg to 3.8.10¹¹ vg	40 μL/deposit	3 μL/min	AAV vector was detected in a brain volume of 12–15 cm³ that corresponded to 37–46% of the injected hemisphere. ARSA enzyme was expressed in multiple interconnected brain areas over a distance of 22–33 mm. ARSA activity was increased by 12–38% in a brain volume that corresponded to 50–65% of injected hemisphere	³⁷
GDNF for PD	Striatum, substantia nigra, caudate nucleus	NHP, rhesus	Eight aged (25 years) + five young adults	8 + 5	LV				Extensive GDNF expression with anterograde and retrograde transport was seen in all animals. In aged monkeys, lenti-GDNF augmented dopaminergic function. In MPTP-treated monkeys, lenti-GDNF reversed functional deficits and completely prevented nigrostriatal degeneration. Additionally, lenti-GDNF injections to intact rhesus monkeys revealed long-term gene expression (8 months). In MPTP-treated monkeys, lenti-GDNF treatment reversed motor deficits in a hand-reach task	⁷⁴
APOE2-HA for AD	Intrahippocampal	NHP	4- to 7-years old	2	Recombinant AAVrh.10	5.10¹² vg (vg; 0.7–1.2.10¹² vg/kg)	15 μL per injection site	1 μL/min	AAVrh.10hAPOE2-HA directly into the hippocampus/entorhinal cortex achieved easily detectable, diffuse ApoE2 expression in targeted regions using this route of delivery compared with the nontreated controls	⁸²
NAGLU for lysosomal disease	White matter	Dog		25	AAV2.5 AAV5.5	5.10¹¹ vg, 1.5.10¹² vg/mL; 8.10¹¹ vg, 2.5.10¹² vg/mL; 20.10¹¹ vg, 6.5.10¹² vg/mL	8 × 40 μL	2 μL/min	In immunosuppressed dogs, vector was efficiently delivered throughout the brain, induced α-N-acetyl-glucosaminidase production, cleared stored compounds and storage lesions	¹⁸³
CLN2 for late infantile neuronal ceroid lipofuscinosis	Head and body of the caudate nucleus, hippocampus and overlying cerebral cortex	NHP			AAV2	3.6 × 10¹¹ pu	180 μL	1 μL/min	Assessment at 5 and 13 weeks demonstrated widespread detection of TPP-I in neurons, but not glial cells, at all regions of injection. The distribution of TPP-I-positive cells was similar between the two time points at all injection sites	¹⁸⁴
α-Iduronidase	Putamen and centrum semiovale	NHP		6	rAAV2/1, rAAV2/2, and rAAV2/5	1.4.10¹⁰ vg	2 × 50 μL		Global diffusion throughout the brain was not significantly different between the three serotypes. However, rAAV2/1 and rAAV2/5 resulted in higher vector copy numbers per cell than did rAAV2/2, respectively, in the brain and the distal neuronal structures	¹⁸⁵
hrGFP	Corona radiata, striatum, and basal forebrain	NHP cynomolgus		8	AAV1	2.3–6.9.10¹¹ vg	10–150 μL	0.2–3 μL/min	AAV1 is actively trafficked to regions distal from the infusion site. In addition to neuronal transduction, a significant non-neuronal cell population was transduced by AAV1 vector	¹⁸⁶
AAV-TK, AAV2-AADC		NHP			AAV2				At least 75% of the putamen could be covered by a single infusion of the vector	¹⁸⁷
Human acidic sphingomyelinase For Nieman Pick disease	Brainstem (one site) and thalamus (bilateral)	NHP		4	AAV2	1.10¹² vg/mL	33–199 μL	0.1 increased at 10-min intervals to 0.2, 0.5, 0.8, 1.0, and 2.0 μL/min	We found that enzymatic augmentation in brainstem, thalamic, cortical, as well subcortical areas provided convincing evidence that much of the large NHP brain can be transduced with as few as three injection sites	¹⁸⁸
hARSA	External capsule and thalamus	NHP	2–3 months	9	Lentivirus	5.10⁷ TU/injection site	80 μL	NA	Favorable safety profile and consistent pattern of LV transduction and enzyme biodistribution. Efficient gene transfer in neurons, astrocytes, and oligodendrocytes close to the injection sites resulted in robust production and extensive spreading of transgenic enzymes in the whole CNS	¹⁸⁹
Heparan sulfate proteoglycan receptor	Thalamus+ICV	NHP			AAV2				The combination of thalamic and ICV delivery resulted in transduction of oligodendrocytes in superficial cortical layers and neurons in deeper cortical layers	¹⁹⁰
hARSA	White matter, six sites	NHP		14	AAVrh10	5.5.10¹¹ vg/hemisphere	30 μL for each deposit, 0.5 μL/min	0.5 μL/min	After injection of the 1 ·dose, AAVrh.10-hARSA vector was detected in a large part of the injected hemisphere, while ARSA activity exceeded the normal endogenous activity level by 14–31%	¹¹
hASM for NP-A	Twelve sites: motor cortex, occipital cortex, striatum and thalamus, hippocampus, cerebellum	NHP	2 years old	2	AAV1	2.6.10¹² vg	520 μL total	NA	A combination cortical, subcortical, and cerebellar injection protocol could provide therapeutic levels of hASM to regions of the NHP brain	¹⁹¹
ARSA	Six sites deep gray matter+white matter	NHP	3- to 6-years old	11	AAVrh.10	1.5.10¹² vg	50 μL/deposit	1–3 μL/min	Of the five routes studied, administration to the white matter generated the broadest distribution of ARSA, with 80% of the brain displaying more than a therapeutic (10%) increase in ARSA activity above PBS controls	⁸¹
SGSH	Subcortical white matter	Dogs		3	AAVrh.10	1.10¹² vg and 2.10¹² vg	500 μL/deposit (2 or 4 deposits)	10 μL/min	Extensive distribution into both rostral and caudal brain regions. significant amounts of vector DNA were found in only 37% of brain punches, increases of SGSH activity of 20% or greater relative to vehicle-treated animals were found in 78% of the brain punches tested 4 weeks after injection	⁷³
SGSH	Subcortical white matter	NHP	4 Years	2	AAVrh.10	7.2.10¹¹ vg	50 μL/deposit (four deposits)	5 μL/min	Presence of vector DNA in a limited proportion (11%) of brain punches, but a wide distribution of SGSH enzymatic activity of 20% or more of control levels in the near totality (97%) of the NHP brain 6 weeks after injection	⁷³
	Striatum	NHP			Tricistronic LV				Restoration of extracellular concentrations of dopamine and corrected the motor deficits for 12 months without associated dyskinesias	⁷⁵

AAV, Adeno-associated viral; AD, Alzheimer's disease; CNS, central nervous system; ICV, intracerebroventricular; LV, lentiviral vector; MLD, metachromatic leukodystrophy; NHP, nonhuman primate; PBS, phosphate-buffered saline; PD, Parkinson's disease.

Recent studies suggest that AAV5 and AAVrh10 have more global transduction with widespread distribution in the brain^11,72,73 and spinal cord. LVs are able to deliver the therapeutic gene in a restricted area.^74,75

Intracerebrospinal fluid delivery

As an alternative to intraparenchymal delivery and to target larger brain or spinal cord volumes, especially in neurodegenerative diseases involving widespread regions, intra-CSF delivery is an option.

For this purpose, three major routes can be used: ICV delivery into the ventricles, the cisterna magna as widely used in large animal models (Table 1), or by IT delivery into the CSF surrounding the spinal cord (Table 2).

Table 2.

Intracerebroventricular administration in large animal models

Transgene/Pathology	Target	Specie	Age	Number	Vector/Buffer	Dose	Volume	Speed	Results	Article
shRNA SMN+GFP or SMN	Cisterna magna	Pig	5 days	14	scAAV9	6.5.10¹² vg/kg (shSMN)		Slow and constant	Reduction of SMN mRNA levels by 73% in motor neurons postnatally	⁷⁷
GFP	Cisterna magna	NHP	Adult	3	AAV9	1.1.10¹³ vg	2 mL		Presence of serious adverse side effects with a nonpronounced cellular loss in the Purkinje layer. No side effect in animal with more restricted GFP expression	⁸⁰
hAADC	Cisterna magna	NHP	Adult	1	AAV9	3.10¹³ vg	2 mL		Broad transduction throughout cerebellum and brain cortex similar to what was obtained with GFP but no cerebellar cell dysfunction.	⁸⁰
GFP	Cisterna magna	Sheep	Adult	3	LV	1.3.10⁸ TU (N = 2) and 1.10⁹ TU	10 and 100 μL	1 min	GFP expression in cells along the needle track and in the brain parenchyma up to 2.5 mm rostral-caudal and lateral to the injection site. Both neurons and astrocytes were similarly transduced	⁷⁶
VEGF	Cisterna magna	Cat	Adult	3	ssAAV1	10¹²/kg	1 mL/kg		Strong expression of the transgene in the spinal cord, even at the lowest dose	¹⁹²
VEGF	Cisterna magna	Cat	Adult	4	scAAV9	10¹²/kg and 10¹³/kg	1 mL/kg		Numerous GFP-positive neurons in the cortex, thalamus, and cerebellum	¹⁹²
GFP	Cisterna magna	NHP	Adult	2	AAV7 (saline, 5% sorbitol, 0.001% Pluronic F-68)	2.10¹³ vg	2 mL	0.5 μL/min	Cells throughout the NHP brain from prefrontal to occipital cortex and cerebellum, sparsely in striatum. GFP-positive cells clustered around blood vessels weak transduction in periphery. GFP-positive cells at all spinal cord levels, in motor neurons and some astrocytes DRG of the cervical region, proximal to the site of injection and some satellite glial cells surrounding DRG neurons	⁶⁷
GFP	Cisterna magna	NHP	Adult	2	AAV9 (saline, 5% sorbitol, 0.001% Pluronic F-68)	1.8.10¹³ vg	2 mL	0.5 μL/min	Cells throughout the brain from prefrontal to occipital cortex and cerebellum, sparsely in striatum. GFP-positive cells clustered around blood vessels. GFP-positive cells at all spinal cord levels, in motor neurons and some astrocytes. DRG of the cervical region, proximal to the site of injection and some satellite glial cells surrounding DRG neurons	⁶⁷
GFP	Cisterna magna	NHP	2–3 years	3	sc AAV9	1.25.10¹³ vg	3 + 3 mL		15–50% Motor neuron transduction throughout the cervical, thoracic, and lumbar segments. GFP-positive pyramidal neurons; Purkinje cells throughout the cerebellar cortex were also efficiently transduced. Not detected in the liver and spleen of the monkeys	⁸³
GFP	Cisterna magna	Dog	Adult	7	AAV9	2.10¹³ vg	1 mL	100 μL/min or bolus in 15 s	Widespread CNS transduction	⁸⁵
Human SGSH	Cisterna magna	Dog	Adult	2	AAV9	2.10¹³ vg	1 mL	Bolus in 15 s	The loss of transgene expression was due to the use of a nonspecies-specific transgene
Canine SGSH	Cisterna magna	Dog	Adult	3	AAV9	2.10¹³ vg	1 mL	Bolus in 15 s	Activity peaked 2–4 weeks, but persistent at high levels for 3 months
eGFP	Cisterna magna	NHP	Adult	4	ssAAV9	5.10¹²/kg and 2.5.10¹²/kg			Substantial vector deposition in the brain and spinal cord (1 VGC) Vector copy numbers quite high in liver and spleen	⁸⁴
ApoE2/HA	Cisterna magna	NHP	Adult	3	AAVrh10	5.10¹³ vg	1–1.3 mL	0.5 mL/min	CM: 92.0% of cubes had vector levels >1,000 copies/lg DNA, heavy staining of the ependymal cells of the choroid plexus, but also in areas around the frontal and mid-brain, including the hippocampal region, as well as areas around the posterior of the brain and spinal cord	⁸²
GFP	Cisterna magna	NHP	Adult	2	AAV9 sc	1.8.10¹³ vg	2 mL	0.5 μL/min	Prefrontal to occipital cortex and mainly cerebellum. Number and intensity of GFP-positive cells were much greater after CM infusion than ICV delivery. Greater astrocytic than neuronal tropism via both routes. Not shield against AAV antibodies. Sparse GFP expression was observed in the spleen and liver	⁷⁹
hAADC	Cisterna magna	NHP	Adult	5	AAV9	0.3/1/3.10¹³ vg	2 mL	0.5 μL/min	Prefrontal to occipital cortex and mainly cerebellum. Number and intensity of GFP-positive cells were much greater after CM infusion than ICV delivery, greater astrocytic than neuronal tropism via both routes. Not shield against AAV antibodies. Sparse GFP expression was observed in the spleen and liver	⁷⁹
GUSB	Cisterna magna	Dogs	Adult and 2 months	7	AAV9	1.8.10¹³ vg	1 mL	1–2 min	ICV and IC vector administration resulted in similarly efficient transduction throughout the brain and spinal cord. ICV cohort developed encephalitis associated with a T-cell response to the transgene product, a phenomenon that was not observed in the IC cohort	⁹⁵
GFP	Cisterna magna	NHP	Adult	2	AAV9	1.83.10¹² vg	1.5 mL		Substantial transduction was found in the hypothalamus along the third ventricle and in the central gray surrounding the Sylvian aqueduct. Extensive transduction was found in the subcommissural organ, located within the dorsal third ventricle, whereas some (GFP)-positive vestibular neurons were found near the fourth ventricle	⁸⁶
GFP	Cisterna magna	NHP	Adult	2	AAV2, 5	2.10¹² vg	1.5 mL		Very strong transduction of the ependymal structure	⁸⁶
GFP	Cisterna magna	Cat	50 days	6	scAAV9	10¹² vg/kg	1–1.5 mL		More efficiently targeting of MNs of the lumbar (99%) than of the cervical regions (84%) spinal cord. Numerous oligodendrocytes were also transduced in the brain and in the spinal cord white matter of young cats, but not of neonates	⁸⁸
ARSA-FLAG/MLD	Lateral ventricle	NHP	Adult	2	AAVrh10 in PBS	1.5.10¹² vg	75 μL	15 μL/min	Almost no copies and no ARSA expression (11/83 = 13%) whereas gold standard: white matter 82%	⁸¹
ApoE2/HA	Left or right ventricle	NHP	Adult	3	AAVrh10	5.10¹³ vg	1–1.3 mL	0.2 mL/min	ICV: 90.0% of cubes had vector levels >1,000 copies/lg DNA, heavy staining of the ependymal cells of the choroid plexus, but also in areas around the frontal and mid-brain, including the hippocampal region, as well as areas around the posterior of the brain and spinal cor.	⁸²
TPP1/CLN2	Lateral ventricles±CM	Dog (TPP deficient)	10–11 days	7	AAV2	1.1.10¹² vg	1.5 mL		TPP1 activity in CSF detectable at 5 days but no more detectable at 2 months	⁷⁸
IDUA/MPS I	Suboccipital	Cat	Adult	5	AAV9	10¹² vg/kg	1–2 mL	Bolus	Complete correction of biochemical and histological manifestations throughout the CNS. Antibody responses against IDUA, which reduced detectable enzyme without substantially reducing efficacy. No evidence of toxicity.	⁸⁴
GFP	ICV	Dog	Adult	2	AAV9	2.10¹³ vg	1 mL	Bolus in 15 s	Comparable with CM administration	⁸⁵
GFP	ICV	NHP			AAV2—HBKO	1.8.10¹³ vg	1.5 mL	1–10 μL/min	Widespread cortical transduction, with oligodendrocytes transduced. Robust motor neuron transduction observed in all levels of the spinal cord	¹⁹⁰
hASM-HA	CM	NHP	Adult	6	AAV9	1.32.10¹³ vg	6 mL	1 mL/min (n = 3) or 1 mL/h (n = 3)	Infusion of the vector in brain and spinal cord after MRI but only with High-speed delivery	¹⁹³
hASM-HA	ICV	NHP	Adult	2	AAV9	1.32.10¹³ vg	6 mL	1 mL/min (n = 3) or 1 mL/h (n = 3)	Much larger cortical distribution at the end of the acquisitions, including occipital cortical regions that were not covered by any other routes of delivery	¹⁹³
cIDUA	Suboccipital	Dogs	28 days	3	AAV9	10¹² vg/kg	0.5 mL	Bolus	Vector distributed throughout the CNS. Supraphysiologic expression of IDUA in CSF, which declined to the normal range	⁹⁰

CM, cisterna magna; CSF, cerebrospinal fluid; DRG, dorsal root ganglia; IC, intracisternal; MRI, magnetic resonance imaging; mRNA, messenger RNA; shRNA, short hairpin RNA; SMN, survival motoneuron protein; VGC, vector genome copy.

LVs have been used in one approach in adult sheep, but this reported attempt resulted in very limited transduction close to the needle track with up to 2.5 mm rostrocaudal transduction.⁷⁶ AAVs lead to greater transduction especially in adults. ICV delivery in young animals differ, providing extensive transduction of motor neurons in some cases. Using scAAV9 in 5-day-old pigs⁷⁷ and in 10- to 11-day-old dogs, Katz et al. reported a TPP1 activity increase in CSF a few days after transduction, but then loss of the transgene owing to immune response against the transgene product.⁷⁸ AAV9 and AAVrh10 have been predominantly injected with doses ranging from 10¹² vg to 5.10¹³ vg in dogs, NHP, and cat^{67,79

–87} mainly in the cisterna magna with either GFP or therapeutic genes (Table 2). Diffuse transgene expression was obtained in the cortex and cerebellum (Table 2). In most studies, efficacy of spinal cord targeting was not analyzed; however, available studies report highly variable results with large motor neuron transduction.⁸⁸

As an alternative to ICV delivery or depending on the pathology to improve spinal cord targeting, delivery in the CSF can be achieved through IT injection. Studies have been performed in dogs,^85,89,90 NHP,^13,84,91 pigs,^83,92 and marmosets⁹³ mainly using AAV9 or AAVrh10 vector in neonates (5 days old) or in adult animals (Table 3). Two major techniques are routinely used for IT delivery, either a single delivery without the use of a catheter, generally performed in the L4/L5 space, or with prior insertion of a catheter to allow single or multiple deliveries.^83,94 IT delivery leads to efficient motor neurons transduction ranging from 10–30%⁸³ to 80%¹³ depending on the study (Table 3), but sparse transduction in the brain (Table 3). Two studies have evaluated placing the animal in the Trendelenburg position with the feet elevated above the level of the head to improve the upward diffusion of the vector. Variable results were reported, one showing no improvement⁹⁵ and the other one a slight increase, with up to 55% targeting of cervical motor neurons¹³ (Table 3).

Table 3.

Intrathecal administration in large animal models

Transgene/Pathology	Target	Species	Age	Number	Vector/Buffer	Dose	Volume	Speed	Results	Article
GUSB/MPS VII	IT	Dog	18–20 days	3	AAV9	5.10¹² vg/kg	1–2 mL	1–2 min	∼50-Fold higher expression and a more profound effect on markers of disease than IV delivery	⁸⁹
GUSB/MPS VII	IT	Dog	18–20 days	2	AAV10	5.10¹² vg/kg	1–2 mL	1–2 min	∼50-Fold higher expression and a more profound effect on markers of disease than IV delivery	⁸⁹
GUSB	Lumbar L4/L5	NHP	Adult	6	AAV9	2.10¹³ vg	5 mL		Transduction efficiency was not improved by placing animals in the Trendelenburg position after injection	⁹⁵
GFP	Lumbar cistern	NHP	Adult	8	AAV9	1.83.10¹² vg (N = 6) and 5.5.10¹² vg (N = 2)	1 mL		Very strong transduction of the ependymal structure	¹⁹⁴
GFP	L4/L5	NHP	Adult	7	scAAV9	1.10¹³ vg/kg	1 mL	Bolus	73% of motor neurons targeted in the lumbar region, 53% of motor neurons targeted in thoracic region, and 29% in the cervical spinal cord; tilting for 10 min was sufficient to increase motor neuron transduction to 55%, 62%, and 80% in the cervical, thoracic, and lumbar region. GFP in all brain regions with particularly strong signals in the hippocampus and in the motor cortex. In the brainstem, high transduction of the hypoglossal and trigeminal nuclei, with >60 and 75% motor neurons. In the cerebellum, both Purkinje cells and cells of the nuclear layer demonstrated high GFP expression	¹³
miRNA anti SOD1	L5 and catheter	NHP	Adult	32	AAV10		2.5 mL	7.5 mL/h	Preimplantation of a catheter and placement of the subject with head down at 30° during IT infusion. Efficient delivery and effective silencing of the SOD1 gene in motor neurons	⁹⁴
GFP	L5/L6	Pig	5 Days	1	AAV9	5.2.10¹² vg/kg	0.25 mL	Bolus	Extensive motor neuron transduction at all levels of the spinal cord. The brain regions with the highest levels of GFP expression were cerebellar Purkinje cells, nerve fibers within the medulla and discrete nuclei, such as the olivary nucleus. Expression within the rest of the brain was restricted to scattered cells near the meningeal surfaces. No obvious expression in periphery	⁹²
miRNA anti SOD1	Lumbar	Marmosets	<4 years	9	AAV10	6.10¹² vg/kg	300 μL	Bolus	High transduction of lumbar spinal cord, than thoracic and cervical. High liver transduction. In brain, mild to good transduction, depending on the regions. Robust GFP staining was seen at the injection site at LSC level all the way to CSC level	⁹¹
GFP	L5 with catheters	Pigs	2 months	2	scAAV9	3 × 1.10¹² vg	3 × 0.5 mL	Bolus	GFP expression in 10–30% of the motor neurons, and one segment (L2) showed GFP expression in 35% of the motor neurons	⁸³
eGFP	Lumbar puncture	NHP	Adult	2	ssAAV9	2.5.10¹²/kg			10 × lower gene transfer throughout the spinal cord, and up to 100-fold less in the brain compared with CM. Vector copy numbers quite high in liver and spleen	⁸⁴
hASM-HA	Lumbar	NHP	Adult	3	scAAV9	1.32.10¹³ vg	6 mL	1 mL/min or 1 mL/h	Leakage of the infusion in musculature	¹⁹³
GFP	Lumbar	Sheep	Adult	2	scAAV9		15 mL	1 mL/min	The frontal, occipital, and parietal cortices exhibited extensive neuronal and glial cell transduction, whereas in the motor cortex glial transduction was primarily observed. Scarce positive neurons were present in the caudate, putamen, and thalamus. Strong GFP staining was noted in the cerebellum, including Purkinje cells, deep cerebellar nuclei and adjacent axons in the white matter, cerebellar peduncles, and brainstem. Robust neuronal transduction was observed along the entire length of the spinal cord, from cervical to lumbar	¹⁹⁵
miRNA anti SOD1	Lumbar L4/L5	Marmoset	Adult	1	AAV10	2.7.10¹² vg	250 μL	Slow bolus	Robust expression in motor neurons along the full length of the spinal cord	¹⁹⁶

IV, intravenous.

Even if ICV delivery is designed to target basal ganglia (Fig. 1d), intracisternal delivery to target the cortex (Fig. 1c) and IT delivery for the spinal cord (Fig. 1b), the direct relationship between the site of injection and the efficient delivery to the target has never been proven, and appear to be different between NHP and human in relation to the volume of the respective brains and the distance between CSF pathways and the CNS targets.

In between intraparenchymal and intra-CSF delivery, for intraspinal targets, spinal subpial delivery has been proposed in mice, rats, and pigs.^96,97 Despite the short distances for spinal diseases, the primary issues remain the need for an open microneurosurgical approach after laminectomy (or a laminotomy) and the risk of neurological deficits secondary to spinal cord subpial bleeding.

Intravenous delivery

As an alternative to more invasive injections, IV delivery has recently become an option to target the CNS, especially with the SMA trial.¹² AAV9 and AAVrh10 are once again the major serotypes injected dosing from 10¹² vg/kg up to 10¹⁴ vg/kg. In neonates (2–7 days old) studies have demonstrated up to 39% transduced motor neurons in cats⁷⁷ but other studies were unable to detect the transgene⁸⁸ (Table 4). In adults, it differs with mild to no neuronal transduction^81,90 either in spinal cord and brain (Table 4). Transduction was in any case much lower compared with ICV⁸⁴ or IT delivery and glial cells were predominantly transduced,^86,92 without clear explanation. In addition, IV delivery led to a significant transduction of peripheral organs (Table 4).

Table 4.

Intravenous administration in large animal models

Transgene/Pathology	Target	Specie	Age	Number	Vector/Buffer	Dose	Volume	Speed	Results	Article
GFP	Internal carotid artery	NHP		3	AAV9 sc	3.10¹³ vg	11/21/40 mL	4 mL/min	Broader transgene distribution throughout the CNS, extending through several cortical regions. Scattered GFP-expressing cells were found from the pre- frontal to occipital cortex, and in the cerebellum. Widespread peripheral organ transduction (liver and spleen)	⁷⁹
ARSA-FLAG/MLD	Intra-arterial (right middle cerebral artery)	NHP	Adult	1	AAVrh10 in PBS	1.5.10¹² vg	12 mL	Bolus	Almost no copies and no ARSA expression (6/77 = 7.8%) whereas gold standard: white matter 82%	⁸¹
GUSB/MPS VII	IV	Dog	3 Days	1	AAV9	2.10¹³ vg/kg	1–2 mL	1–2 min	Limited GUSB expression in cortical and hippocampal neurons, and Purkinje cells, while average expression levels in other brain tissues were only ∼1–6% of normal levels, similar to AAV10 injected animals. Clear motor neurons transduction	⁸⁹
GUSB/MPS VII	IV	Dog	3 Days	1	AAV10	2.10¹³ vg/kg	1–2 mL	1–2 min	Limited GUSB expression in cortical and hippocampal neurons, and Purkinje cells, while average expression levels in other brain tissues were only ∼1–6% of normal levels, similar to AAV9 injected animals. Clear motor neurons transduction	⁸⁹
NAGLU	IV	NHP	Adult	8	ssAAV9	1 (N = 2) or 2.10¹³ vg/kg (N = 4)	5 mL	Bolus	Global CNS and broad somatic transduction. Evident vector transduction throughout the brain. Low levels of preexisting anti-AAV9 antibodies did not diminish vector transduction but high-level of preexisting anti-AAV9 Abs lead to reduced transduction in liver and other somatic tissues, but had no detectable impact on transgene expression in the brain	³⁹
GFP	Carotid artery	Cat	2 Months	1	AAV-B1	3.4.10¹² vg		Bolus	Sparse but widespread neuronal gene transfer throughout the brain. AAV-B1 transduced neurons in the cerebral cortex, striatum, hippocampus, thalamus and Purkinje neurons in the cerebellum and motor neurons throughout the midbrain. No indication that AAV-B1 transduced endothelial cells. Negligible transduction of liver was observed with AAV-B1, while strong gene transfer to skeletal and cardiac muscle could be detected	¹⁹⁷
VEGF	Jugular vein	Cat	2 Days	3	scAAV9	10¹² vg/kg	1 mL/kg	Bolus	No detectable protein	⁸⁸
GFP	Jugular vein	Cat LIX1	7 Days	2	scAAV9	1.5.10¹² vg	1 mL	Bolus	GFP was detected from the cervical part of the spinal cord to the cauda equina in a number of cells in the ventral spinal cord. In neonates, up to 39 and 34% of the MNs. Nerve fibers of the fasciculi gracilis and cuneatus dorsal sensory tracts also contained large amounts of GFP	^.87
GFP	Jugular vein	Cat LIX1	7 Weeks	2	scAAV9	1.2.10¹² vg	3.6 mL	Bolus	Up to 15% of MNs transduction observed	^.87
cIDUA	Jugular vein	Dogs	90 Days	4	AAV8	3.10¹² vg/kg	0.5–1 mL	Bolus	Mild in serum IDUA activity	⁹⁰
GFP	Saphenous or intracarotid	NHP	Adult	4	sc AAV9	0.9–1.10¹³ vg/kg	10 mL/kg	2.5 mL/min	Predominant transduction of glia in NHPs after both intravenous and intra-arterial administration	⁸⁶
VEGF	Jugular vein	Cat	Adult	6	scAAV9	10¹²/kg (N = 2) and 10¹³/kg (N = 4)	1–2 mL		Transduction of the spinal cord occurs in neonatal and adult cats but the level of transgene expression remained low in both adult and neonate-injected animals. Transduction was detected essentially in liver	¹⁹²
GFP	Saphenous vein	NHP	Adult	1	AAV9	1–3.10¹⁴ vg/kg		Bolus	Only MN and cells with glial morphology that were sparsely scattered. Most abundant number of GFP-expressing cells in all cortical regions lateral geniculate midbrain, pons and medulla. Subcortical structures such as thalamus and putamen were also GFP+ but at a lower cell density. Mostly glial transduction with microglia and astrocytes. High levels of vector in liver and also other peripheral tissues	⁹²
GFP	Catheter through the brachial artery until aorta	NHP	Adult	1	AAV9	2.7.10¹³ vg/kg		Bolus	Motor neuron targeting extensive transgene expression throughout the entire brain. Most abundant number of GFP-expressing cells in all cortical regions lateral geniculate, midbrain, pons, and medulla. Subcortical structures such as thalamus and putamen were also GFP+ but at a lower cell density. Glial transduction with microglia and astrocytes. Liver, heart, testis and largely peripheral transduction	⁹²
IDUA	IV	NHP	Adult	2	AAV9	2.5.10¹³ vg/kg 7.5.10¹³ vg/kg	1 mL	Bolus	Vector well tolerated. GFP expression was detected in most non-CNS tissues, including liver and muscle, kidney, pancreas, heart, spleen, and pituitary. Very low transduction was observed the frontal cortex and spinal cord and hippocampus and cerebellum but transduction in DRG. Good tolerance of the virus	⁴³
IDUA	IV	NHP	Adult	2	AAVPHP.B	2.5.10¹³ vg/kg 7.5.10¹³ vg/kg	1 mL	Bolus	Vector well tolerated. GFP expression was detected in most non-CNS tissues, including liver and muscle, kidney, pancreas, heart, spleen, and pituitary equivalent to AAV9 except for in skeletal muscle, where transduction was higher for PHP.B vector well tolerated. Immune response against the virus	⁴³
eGFP	IV	NHP		1	ssAAV9	2.10¹³ vg/kg			Vector distribution to the CNS was substantially lower than that achieved at four- to eightfold lower doses via CM	⁸⁴

MN, motoneurons.

Routes of Administration in Clinical Studies

Although IV delivery is readily transferred from animal models to human trial conditions, CSF or brain delivery techniques must be carefully adapted for human conditions (brain anatomy and volumes). ICV, cisternal and IT methods can however all be used in human subjects. Very little intraventricular vector diffusion occurs after cisternal or IT injection; most of the product remains in the spinal compartment or reaches the pericerebral space. Because of the size of the brain and the distance between the ventricles and the cortex, it is impossible to achieve consequent and homogenous diffusion by any CSF route.

Intraparenchymal injection is possible in the human brain, in deep nuclei, or in the white matter (but may need multiple simultaneous injections to obtain sufficient diffusion), but there is no reliable technique for delivering to the spinal cord parenchyma.

The doses of vector delivered depend on the route of administration. Although intraparenchymal delivery requires small amounts of vector ranging from 10⁹ to 2.5.10¹² total vg in NHP and human, doses from 10¹² to 5.10¹³ vg are required for ICV or IT delivery. IV delivery dramatically increases the number of particles required, from 5.10¹² to 5.10¹⁴ vg (10¹² to 10¹⁴ vg/kg) in NHP and close to 10¹⁵ vg in patients in the SMA trial (2.10¹⁴/kg) (Tables 1–6).

Table 6.

Clinical trials with Intra parenchymal administration

Transgene/Pathology	Target	Age	Number	Vector/Buffer	Dose	Volume	Speed	Identification of the Trial and Results if Available	Article
AADC/ParkD	Striatum	Adult		AAV2				NCT00229736, Increased risk of intracranial hemorrhage, phase I motor improvement	¹³⁰
TPP-1/LINCL	12 sites in WM	Children		AAV2				Insufficient but suggestion of slowing progression of disease	¹³⁰
NTN (CERE-120)/Parkinson	Putamen		12	AAV2	1.4.10¹¹ vg/mL and 5.7.10¹¹ vg/mL	40 μL		Well-tolerated, significant motor score improvement 6 months after infusion	¹²⁹
NTN (CERE-120)/Parkinson	Putamen	Adult, 35–75 years	38/20 Sham	AAV2	5.7.10¹¹ vg/mL	40 μL/hemisphere		NCT00252850, CERE 120, double-blinded clinical trial, after 1 year: 13/38, 4/20 sham reacted adversely to the injection, no clinically significant improvement versus placebo at 12 months	¹²⁹
NTN (CERE-120)/Parkinson	Putamen + substantia nigra, bilateral	Adult 35–70 years	60	AAV2	2.4.10¹² vg			NCT00985517, Phase I, double blinded, safely tolerated, 5 years follow up, no significant improvement	¹²⁹
AADC/Parkinson	Putamen		10	AAV2	9.10¹⁰ vg and 3.10¹¹ vg			Low and high vector dose, well-tolerated, dose-dependent improvement in dopamine synthesis; elevated PET signal persisted over 4 years in both groups	¹²⁹
Prosavin	Striatum	Adult, 48–65 years	15	Lentiviral EIAV	1.9.10⁷; 4.10⁷ and 1.10⁸ TU			NCT01856439, phase I/II, safely tolerated, modest effects but patients with higher dose of prosavin required lower dose of dopamine (enhanced dopamine production?)	¹²⁹
NGF (CERE-110)/Alzheimer		Adult 55–80 years	49	AAV2	2.10¹¹ vg			NCT00876863, phase II, inefficient, safely tolerated	¹⁹⁹
CLN2/Batten	( × 12 by 6 bur holes)	Children	10	AAV2	2.5.10¹² vg	150 μL	2 μL/min	NCT00151216, phase I, radiographical changes in 65% of 60 injections sites	¹⁹⁹
GAD65 and GAD67/Parkinson	Subthalamic Nucleus (unilateral)	Adult, 25–75 years	12	AAV2	1.10¹¹ vg/mL; 3.10¹¹ vg/mL and 1.10¹² vg/mL	50 μL		NCT00195143, phase I/II, safe; neuroimaging improvement, clinical improvement at 12 months postinfusion, decrease of STN activity	¹⁹⁹
NTN (CERE-120)/Parkinson			58	AAV2	5.4.10¹¹ vg			NCT00400634, phase II, not effective	¹²⁹
GAD65 and GAD67/Parkinson	Subthalamic Nucleus	Adult, 30–75 years	44	AAV2	1.10¹² vg			NCT00643890, phase II, well tolerated, improvement at 6 months (not greater than DBS)	¹⁹⁸
ASPA/Canavan			21	AAV2	1.10⁹ vg				¹⁹⁸
ASPA Canavan	( × 6)	4–83 years	13	AAV2	9E11 vg			Phase I/II, no long-term adverse event, slowing of disease	¹⁹⁸
CLN2/Batten	WM		16	AAV rh.10	9.10¹¹ vg (n = 6) and 2.85.10¹¹ vg (n = 10)			NCT01161576, phase I/II	NA
CLN2/Batten	WM		8	AAV rh.10	9.10¹¹ vg and 2.85.10¹¹ vg			NCT01414985, phase I/II	NA
NAGLU/MPSIIIB		Child (20–53 months)	4	rAAV2/5	4.10¹² vg			ISRCTN19853672, phase I/II, well-tolerated, induced sustained enzyme production in the brain, best results in the youngest patients	¹⁰
AADC/Parkinson			5	AAV2	9.10¹⁰ vg			Phase I, safely tolerated, modest improvement in motor coordination at 6 months (placebo effect?)	²⁰⁰
AADC/Parkinson			10	AAV2	3.10¹¹ vg			Phase I, safe possibly effective	²⁰¹
SHSH-IRES-SUMF1/MPSIIIA	Twelve sites in WM	Child (32–70 months)	4	AAV rh.10	7.2.10¹¹ vg	60 μL	2 h	NCT01474343, good tolerance, possible but moderate clinical improvement	⁹
AADC/AADC deficiency	Putamen	Child (1.67–8.42 years)	10	AAV2	1.81.10¹¹ vg	80 μL/target	3 μL/min	NCT01395641, phase I/II, motor development improvement in children	³⁵
AADC/AADC deficiency	SN+VTA							NCT02852213, phase I	NA
CAG-NGF/Alzheimer	Nucleus Basalis Meynert	Adult, 50–80 years	10	AAV2				NCT00087789, phase I, safe, biologically effective	²⁰¹
NGF/Alzheimer		Adult >50 years	8					NCT00017940	NA
GDNF	Putamen	Adult	12	AAV2				NCT04167540/Phase 1B	NA
GDNF/Parkinson	( × 16, 4 in cerebellum)	Adult, >18 years	24	AAV2	9.10¹⁰ vg; 3.10¹¹ vg; 9.10¹¹ vg and 3.10¹² vg			NCT01621581, phase I	NA
ARSA/MLD	Twelve sites in WM	Child, 6–60 months	4	AAV rh.10	1.10¹² vg/kg (n = 2) and 4.10¹³ vg/kg (n = 2)	60 μL/site	0.5 μL/min	NCT01801709, phase I/II	NA
SGSH/MPS IIIA	Six sites in WM	Child	20	AAVrh10				NCT03612869, phase III	NA
miHTT/Huntington	Intrastriatal	Adult	26	AAV5	6.10¹² vg and 6.10¹³ vg			NCT04120493, phase I/II	NA
AADC		Adults 40–75 years	42	AAV2	2.5.10¹² vg			NCT03562494, phase II	NA

EIAV, equine infectious anemia virus; WM, white matter.

Local administration of low doses of vector limits biodistribution and the risk of immunogenicity or toxicity owing to AAV capsid or expression of the transgene. The high doses required for intra-CSF and IV administration raise issues of immunogenicity, manufacturability, and final cost.

Translating gene therapy proof of concept in animal models to clinical application in patients requires adapting delivery protocols. Translation may be simple for intra-CSF or IV delivery, based on the weight of the animal or the volume of the brain tissue or of the CSF. Modeling the delivery into the brain parenchyma is more critical for designing a clinical protocol. This should take into consideration the volume of the target region determined by imaging, the sites and the number of injections, the degree of anatomical precision required, the volumes to be injected, and the flow (constant or CED). Translating these parameters from large animal brain to human brain requires specific anatomical adaptations (needle track in particular) and evaluation of feasibility, safety, and efficiency in terms of biodistribution in animal models under conditions as close as possible to the human clinical procedure.

There is no limit for age, and theoretically, it is possible to treat even newborns by all routes of delivery. It is possible to use frameless stereotactic delivery using magnetic or optic neuronavigation. In addition, robotic systems are now available even in the youngest subjects. The youngest child treated by our team with intracerebral delivery (16 targets supra and infratentorial) was 9 months old.⁹

Intracerebrospinal fluid delivery

To overcome the inability of the gene therapy vectors to cross the BBB, direct injection within the CNS compartment has been attempted.⁹⁸ All trials using these routes of administration are given in Table 5. Three main modalities can be discussed.

Table 5.

Clinical trials with intra cerebrospinal fluid or intravenous administration

Transgene/Pathology	Target	Age	Number	Vector/Buffer	Dose	Volume	Speed	Identification of the Trial and Results if Available	Article
CLN6/Batten	IT		13	AAV9	1.5.10¹³ vg			NCT02725580, phase I/II	NA
JeT-GAN/Giant Axonal Neuropathy	IT			AAV9				NCT02362438	¹⁹⁸
NAGLU/MPSIIIB				scAAV9					NA
SGSH/MPSIIIA	IV		9	AAV sc9	5.10¹² vg/kg (n = 3) and 1.10¹³ vg/kg (n = 1)			NCT02716246, phase I/II	NA
SMN type 1/SMA	IV	Child	15 (Cohort 1 n = 3, cohort 2 n = 12)	AAV9	6.7.10¹³ vg/kg cohort 1 and 2.10¹⁴ vg/kg cohort 2			NCT02122952, phase I/II, longer survival, motor improvement	¹²
SMN	IV	Child	6	AAV9				NCT03837184, phase III	NA
SGSH/MPSIIIA	IV		12	scAAV9	3.10¹³ vg/kg			NCT04088734	NA
GLB1/GM1 type I and II	IV	6 Months to 1 year for GM1 type I and 2–12 for GM1 type II	45	AAV9	1.5 to 4.5.10¹³ vg/kg			NCT03952637	NA
hTERT/AD	IV/IT	45 Years and older	5					NCT04133454	NA
HEXA/HEXB Tay sachs	IT	7 and 30 Months old	2	AAVrh8	HEXA vector (0.5 mL, 9.9.10¹² vg/mL) mixed with HEXB (0.413 mL, 1.2.10¹³ vg/mL) to generate a 1:1 equimolar formulation	12 mL	1 mL/min		¹⁹⁵
GBA/Parkinson	ICM	40–75 Years	16		Low and high dose			NCT04127578	NA
GRN/Fronto temporal dementia	ICM	30–80 Years	15		Low, medium and high dose			NCT04408625	NA
IDUA/MPS1	ICM	4 Months and older	5	AAV9	1 and 5.10¹⁰ gc/kg			NCT03580083	NA
IDS/MPS2	ICM	4 Months to 5 years	6	AAV9	1.3 and 6.5.10¹⁰ gc/kg			NCT03566043	NA
CLN3	IT	Children	7	scAAV9	Low or high dose			NCT03770572, phase I/IIa	NA

SMA, spinal muscular atrophy.

Intrathecal lumbar administration with two potential modalities

Direct unique injection by a lumbar tap or through an intraspinal catheter connected to a subcutaneous reservoir. The use of a single injection by lumbar tapping is a nonsurgical procedure. It could be performed, in a medical environment, with local anesthesia. In difficult cases, the use of fluoroscopy or ultrasonography⁹⁹ can facilitate the procedure (or to guide a catheter insertion). The main inconvenience of this technique is the risk of CSF leak in the extradural space at the moment of needle removal and 2 days after the procedure. Consequently, it is impossible to precisely control the quantity of vector administered within the intradural space. The use of atraumatic G22 needle (Sprotte) can minimize this risk.⁹⁹

To avoid this major inconvenience, an alternative is to install a subcutaneous reservoir connected to an intraspinal catheter.⁹⁹ This procedure must be performed in the operation room, under general anesthesia. Injection must be performed 1 or 2 weeks after the initial surgery and the removal of the system at least 1 week after. This technique guarantees control of the injected volume, but needs two general anesthesia, and increases the risk of infection. The main incertitudes of this technique are determining the exact distribution of the product between the different compartments; intraspinal CSF, intraventricular, and intracranial subarachnoid spaces (Fig. 1). The main disadvantage of the ITL access results from the natural flow of CSF from the intraventricular choroid plexus to the subarachnoid space around the spinal cord and finally to the pericerebral spaces for resorption through the venous system. Reaching the brain target through the CSF stream requires a large dose volume. Other associated strategies can be used to improve the efficacy such as a buffer flush or a Trendelenburg position. Complications owing to the device and its implantation also have to be considered. Meningitis can be a severe infectious complication that needs removal of the system and adapted antibiotics treatment. Other mechanical complication can be encountered such as migration, rupture, or disconnection, kinked or obstructed catheter. A CSF leak around the catheter to the subcutaneous space can lead to an artificial meningocele that can impair the ability to infuse the treatment. To overcome this problem, two other modes of administration have been proposed.

Intracisternal administration^100,101

In most cases, it is possible to inject within the cisterna magna at the level of the craniovertebral junction. Even if it is possible to do it while the patient is awake, general anesthesia will be preferred, especially in the pediatric population. The risk of leakage through this route is very low and in most cases the reservoir is useless.

Intraventricular administration

A third route to the CSF space is to inject directly within the cerebral ventricles. In most cases, it will be carried out in the frontal horn by direct puncture under stereotactic guidance or more frequently now under frameless neuronavigation. In adults, it is easily performed under local anesthesia and light sedation, in the pediatric population general anesthesia is preferable. Installation of an intraventricular catheter connected to a reservoir may be preferred; however, for a single injection, direct injection is safe and efficient. It is also better for controlling the volume of injection and its distribution. However, because of the risk of parenchymal bleeding, most authors prefer the cisternal approach.

Intraparenchymal delivery

Few human trials using the intracerebral route are reported (Table 6), both in adults (Huntington, Parkinson, and Alzheimer diseases) and children (SMA, Canavan, Batten, mucopolysaccharidosis, and MLD). The vast majority of the trials have used AAV vectors (from 10⁷ to 10¹³ vg) with injections in the deep gray nuclei or in the white matter (only for metabolic diseases in children). Most published trials are phase 1/2 and one is phase 3. Several studies obtained promising results even if partial or preliminary. Of importance, few severe adverse events have been reported.

Technical Aspects: State of the Art

Regarding delivery technique, we focus here on intracerebral delivery, indeed, other routes of delivery have been largely described previously and have not been researched as intensively.

Devices for intraparenchymal delivery of therapeutics agents

Intraparenchymal drug delivery systems offer a practical method for bypassing the BBB to deliver gene therapy. Direct access to parenchyma allows delivery at doses and concentrations that would otherwise correspond to very high levels and volumes systemically. However, this method still has constraints and limitations. For >10 years, considerable research has focused on developing methods to enhance drug delivery,¹⁰² through dedicated intraparenchymal devices.

Principles

Intraparenchymal delivery has two principle challenges: minimize backflow along the injection device and promote optimal drug distribution,¹⁰³ often in a spherical tissue volume. The distribution volume depends on infusion flow rate, infusion volume, and number of injection sites and the type of vector that is injected.¹⁰⁴ The occurrence of backflow depends on several variables including cannula radius, infuse flow rate, and tip location.¹⁰⁵ Indeed, many parameters influence the safety and efficiency of infusion: infusion flow rate, cannula size, infusion volume, and drug molecular size/charge.¹⁰⁶ Variations in flow rate impact the location of infusate distribution. Lower infusion rates (<1 μL/min) are associated with distribution localized primarily to the target tissue (they are used for focal injection in specific areas, mainly in gray matter nuclei), whereas higher infusion rates result in increased distribution into the surrounding parenchyma (“overflow”).^103,106 Several techniques have been proposed to increase infusion rates (up to 10 μL/min); among them the use of CED¹⁰⁷ appears to provide the best compromise for extensive diffusion with minimal local damage. They can be used in combination with multiple injections (up to 16)⁹ when the whole brain must be treated (lysosomal diseases) (Fig. 2).¹¹ Increases in infusion rates raise the local pressure around the infusion site and also increases the extent of backflow. Cannula size seems to have no effect on distribution; however, larger cannulas cause more tissue damages and therefore produce decreased resistance pathways along the brain parenchyma–catheter interface that are associated with increased rates of backflow.¹⁰⁶

Figure 2.

Intracerebral injection in NHP and humans. (a) Personal data of intracerebral gene therapy with 12 intracerebral delivery in white matter for San Filippo A and MLD. (b) Neuronavigation software for preplanning. (c) Delivery improvement with MRI Smartflow catheters; (d) immediate postoperative MRI; and (e) brain delivery in NHP with the same MRI Smartflow catheters. NHP, nonhuman primate.

Catheters subtypes

Three kind of delivery devices can be distinguished: catheters derived from another use (especially intraventricular devices), “homemade” designed catheters^9,11,102,108 from teams experimenting with preclinical intracerebral drug delivery or for early clinical studies,⁹ and more recently commercialized catheter for specific intraparenchymal use (often developed from the homemade devices, and for intracerebral chemotherapy). Principal available devices are described in Table 7.

Table 7.

Devices for intracerebral gene therapy delivery

Intracerebral catheter	Developing Team/Company		Back Flow Reduction	Design	Article
Hamilton Syringe	Hamilton	Rigid, steel	No	End port
UCSF “homemade”	UCSF, Medgenesis Therapeutics and Brainlab^® AG	Rigid, fused silica, then flexible canna with rigid tip	Stepped profile	End port, step design	²⁰²
Smartflow	MRI Interventions, Inc/UCSF	Rigid, ceramic, fused silica liner and a polymer sheath	Stepped profile	End port, step design	²⁰³
MEMS	Alycone, Inc., Cornell	Microfrabricated silicon cannula	Stepped profile	Dual lumen, coupled to multiple proximal tubing	²⁰⁴
Neuroinfuse	Renishaw^®/Univ Bristol Renishaw Medical Solutions, Neurological Applications Department, New Mills, Wotton-Under-Edge, Gloucestershire GL12 8JR, UK	Carbothane	Stepped profile/recesses stepped	Sub 1 mm diameter catheters and guide tubes	^205,206
Gliasite/Emory University	IsoRay/Emory University	Inflatable	No	Balloon tipped, 2, 3, and 4 cm with corresponding full volumes of 5, 15 and 35 cc	²⁰⁷
Hollow fiber	Twin Star Medical			Microporous
Cleveland Multiport Catheter	Cleveland Clinic, Infuseon Therapeutics	Rigid	Yes	Multichannel	²⁰¹
Custom-made, Bristol University	Bristol University	Rigid	No	One port	²⁰⁸
Barium—impregnated one port infusion catheter	Vygon Valley Forge, PA			One port	²⁰¹
Barium—impregnated one port infusion catheter	Medtronic^®		Barium impregnated	One port	²⁰¹

Ventricular catheters (2–3 mm outer diameter) that have been implanted in clinical trials to treat glioma have failed to distribute effectively and have been linked to poor distributions.^109,110 Microcatheters (<1 mm outer diameter) seem more reliable, and all FDA-approved catheters for CED belong to this category.

Design

Recent catheters have been designed to reach the goals of parenchyma delivery: minimize invasiveness through a minimal diameter tubing (Casanova), optimize infusion parameters to maximize distribution volume, and a reflux inhibiting feature required to halt backflow along the catheter entry track (Fig. 2).¹⁰⁹ All these characteristics are summarized in Table 7. Means to minimize backflow are the following: polymer-impregnated tips,^102,111 stepped-design cannula,^105,109,110 and recessed-design cannula^109,112 (Bristol). Means to enhance delivery are as follows: multiporous cannula, multiport catheter, mobile-tip catheter, and balloon-type cannula. A “valve tip” has been proposed to prevent blockage by occluding the inner bore of the cannula with a stylet during insertion.¹⁰³ Another new advance is a multisite delivery catheter with one single tube¹¹³ (cleveland multiport cetheter) that allows multiple targeting points and more homogenous delivery in a three-dimensional array.

Pumps and syringes

To enable injection of small volumes at precise low speeds, specific pumps and syringes must be used. For pumps, most are manufactured for research use (Harvard Apparatus, Holliston, MA), and special authorizations must be obtained for clinical use.

Injection systems: combining precision delivery and limited procedure time.

Classical stereotactic techniques are well adapted for injection in a unique site of brain parenchyma—however, new techniques allowing multiple brain injections of the gene therapy for many indications are needed. Specific three-dimensional magnetic resonnance sequences allow precise targeting in white matter or in deep gray matter nuclei (striatum, thalamus, caudate nucleus, and so on). Preplanning using neuronavigation software (Medtronic^® or Brainlab^®) facilitates the choice of trajectories, modeling delivery and rehearsing the surgical procedure. Optic or magnetic neuronavigation system with or without robotic tools (Rosa^®, Renishaw^®, Medtronic, Brainlab) (Fig. 2) allows frameless insertion of multiple cannulas, supra or infratentorially according to the preplanning. Altogether these systems allow the delivery of therapeutic product reproducibly with high precision (∼1 mm). Intraoperative real-time magnetic resonnance imaging (MRI) has also been proposed as an additive tool to verify the position of the cannula and to check the diffusion of the product with simultaneous gadolinium injection,^66,114 although with the risk of long-term toxicity.

Risks Assessment

Linked to the delivery procedure

Risks owing to anesthesia are mainly correlated to the disease itself and related comorbidities. All injections or catheter placement can result in a CNS injury or hemorrhagic complications. Lumbar puncture is at very low risk of complication if carried out at the low lumbar level without spinal cord anomalies such as low tethered cord. Epidural hematoma can occur, especially with repeated punctures. Spinal epidural hematoma can remain asymptomatic but sometimes cause radicular pain and even rare motor impairment or sphincter dysfunction in case of cauda equina syndrome. A motor impairment requires surgical evacuation. Lumbar puncture can cause subarachnoid bleeding leading to radiculitis, pain, and headache. In case of spinal deformity, fluoroscopy or ultrasonography may help to guide the puncture.

Intrathecal catheter placement needs the use of a guide wire to conduct the catheter to the thoracic level or higher and can result in a spinal cord injury and cause motor or sensitive (transient in the vast majority of the cases) dysfunction. The risk of a hemorrhagic complication in the epidural or intradural space is higher in relation to the larger size of the needle.

Intracranial hypotension symptoms can occur secondary to a lumbar puncture and cause severe orthostatic headaches that can be managed with supine position, painkillers, and if needed, blood transfusion.

Intracisternal puncture requires an adequate cisternal space (confirmed by MRI), a motionless situation and an experienced operator. The size of the cisterna magna and the shape of the skull, a Chiari anomaly, or a foramen magnum stenosis increase the risk of neurological complication (Bulbomedullary junction injury responsible for cardiorespiratory arrest).

Intraventricular access is performed through a right frontal trajectory. Bleeding can occur along the trajectory at each level: subcutaneous hematoma or epidural hematoma is rare. Subdural hematoma can occur by direct bleeding or an intracranial hypotension secondary to a CSF loss; intraparenchymal hematoma can be asymptomatic but may, if extended, lead to motor or cognitive impairment. Intraventricular bleeding may be responsible for headaches and sometimes secondary hydrocephalus.

Linked to the treatment

Gene therapy using viral vectors is mainly hampered by immunogenicity^115

–119 particularly with AAV. Indeed, many people have already been infected by wild-type AAV once in their life, inducing anti-capsid neutralizing antibodies (NAb) spread among various serotypes.^120

–123 Moreover, cross-reactive immunologic material (CRIM) status is also important to predict clinical response. CRIM-negative patients with null mutations or out-of-frame stop codons are completely unable to produce protein (that has to be supplied/involved in their disease) and are therefore more predisposed to develop an anti-transgene response.^124,125 Such host immune responses, particularly happening when IV and intramuscular injections are performed, significantly impair delivery of the therapeutic protein and be possibly deleterious for patients.^126
–128 Delivery of AAV vectors directly into the brain induce low or absent anticapsid or anti-transgene NAb titers in serum.^{114,129

–132} Thus, it must be considered for patient inclusion criteria and study design in clinical trials. Conventional strategies to prevent immunogenicity include corticosteroids or immunosuppressive drugs. Corticosteroids (e.g., methylprednisolone and prednisone) and immunosuppressive drugs (e.g., sirolimus, tacrolimus, rituximab, bortezomib, mycophenolate, and cyclosporine) are administered postinjection and eventually in pretreatment in several clinical trials,^12,133,134 although their effects are controversial.^135,136 Other approaches are emerging such as plasmapheresis,^135,136 editing AAV capsids to eradicate epitopes that induce NAb generation,¹³⁷ using tolerogenic nanoparticles,^138,139 and the incorporation in the transgene of microRNAs (miR) target sequences integrated in the expression cassette that specifically repress translation in antigen-presenting cells^140,141 or oligonucleotide sequences that inhibit Toll-like receptor 9 (TLR9) activation (in development by George Church's Laboratory, Harvard Medical School, patent WO2017214378A1).

Future Development and Perspectives

New AAV serotypes with broad CNS transduction after IV delivery

Over the past decade, significant efforts have been expended in updating the natural repertoire of viral vectors and engineering new serotypes.¹⁴² Using the Cre recombination-based AAV-targeted evolution (CREATE) technique, new AAV variants have been isolated, some able to homogenously transduce the CNS, especially neurons and astrocytes, after IV injection in mice,^42,143 and potentially in NHP, although clear evidence of efficacy remain to be demonstrated. Improved CNS transduction is linked to Ly6, which is not expressed in all mouse lines and not in NHP.^144,145 Indeed, a major challenge is to identify capsids that will be able to efficiently pass the BBB and in mice, NHPs, and human subjects. In addition, the selected capsids should be compatible for large-scale production.

BBB opening as a solution to enhance CNS targeting

Temporary disruption of the BBB might help delivery in the brain parenchyma. Osmotic disruption of the BBB with intra-arterial injection of mannitol has been widely studied and allows for delivery of a variety of drugs and agents into the CNS, including viral vectors.¹⁴⁶ However, this technique induces a diffuse opening of the BBB, precluding targeted delivery of drugs, and is potentially associated with significant neurological side effects.

Ultrasound (US)-induced transitory disruption of the BBB is another technique that has gained increasing interest since Hynynen et al. demonstrated that the IV injection of preformed gas bubbles before low-intensity pulsed US sonications allowed for a reduction of the acoustic pressure necessary to safely open the BBB in rabbits.¹⁴⁷ The interaction between US and injected microbubbles is essential for opening the BBB, and mechanisms of BBB disruption may include transcytosis, cell fenestrations, and opening of tight junctions.¹⁴⁸ US-induced BBB disruption can be monitored with MRI, as a contrast enhancement in T1-weighted sequences after gadolinium injection¹⁴⁷ and is limited to the US beam.¹⁴⁹

The safety of the technique has been assessed through preclinical studies. With optimized parameters, histological side effects are limited to red blood cell extravasation and petechial bleeding¹⁵⁰ after both single or multiple US sessions.¹⁵¹ BBB disruption may induce a transitory sterile inflammatory response.¹⁵² Recent studies have confirmed that the technique was clinically well tolerated in NHPs.¹⁵³

This technique was used to deliver an AAV1/2 viral vector in a targeted manner to the striatum of rats. Transduction observed was mainly restricted to neurons and stable for >1 year.¹⁵⁴ Two main approaches have been developed to bypass the skull interface that induces attenuation and distortion of the US beam. In one case, InSightec (Haifa, Israel) developed a 512-element phased-array transducer, the ExAblate^® 4000 system, that allows a transcranial and noninvasive opening of the BBB.¹⁵⁵ On-going clinical trials are currently evaluating the safety of the ExAblate system for drug delivery after BBB disruption (NCT02343991 and NCT02253212). In another case, CarThera SAS (Paris, France) designed an implantable device, the SonoCloud^® device, which can be plugged into the skull and activated through a transdermal needle.¹⁴⁹ Interim results of the first clinical trial (SONOCLOUD, NCT02253212) evaluating the safety of this system in adult patients treated for recurrent glioblastoma with systemic carboplatin have shown no dose-limiting toxicities and no treatment-related serious adverse events.¹⁵⁶ A phase I trial (SONOKID) assessing the safety of repeated BBB disruptions by the SonoCloud device in association with IV chemotherapy in recurrent supratentorial malignant primitive tumors in the pediatric population was launched in 2020.¹⁵⁷ More recent studies have been reported with percutaneous US but with no additional value.¹⁵⁸

Encapsulated cells and optogenetics

Encapsulated cell technology (ECT) eventually combined with optogenetics allows the delivery of treatment in a continuous or in a controlled/discontinuous ways, respectively.

ECT is a concept based on the confinement of the grafted cells within a permeable device.

ECT has already been shown to be well tolerated in large animals such as dogs for the treatment of intervertebral disc degeneration¹⁵⁹ and in pigs¹⁶⁰ and NHPs¹⁶¹ for AD. Moreover, ECT has already been translated to clinical studies where the safety, feasibility, and tolerability of procedure has been shown, for example, the delivery of neurotrophic factor in disorders of eye^162
–164 (clinicalTrial.gov NCT00447980, NCT00447993, NCT02228304, and NCT00447954), in AD¹⁶⁵ (NCT01163825), in Huntington's disease,^166,167 and in amyotrophic lateral sclerosis.¹⁶⁸ Moreover delivery of anti-amyloid immunotherapy by ECT is described in AD patients.^169,170 Thus, ECT provides an innovative approach for the local and systemic delivery of a recombinant protein in the CNS.

The optogenetic approach uses optical methods to modulate the cellular expression of molecules, which are activated by irradiation with light energy by genetic engineering to control/regulate cellular function or intracellular signal transduction.¹⁷¹ One of the challenges of optogenetics is its translatability to the clinic. Many improvements in optogenetic technologies in NHPs have been performed to exert precise control of specific cells or brain regions at the millisecond timescale and to reliably transduce cells and readout the optically induced neural modulation.^172,173 A new optogenetic approach has been described by Ruiz et al., 2013 in which the native dura is replaced with optically transparent artificial dura allowing visual monitoring of the expression of the optogenetic agent over time.¹⁷⁴

The first optogenetic study of NHP was performed in 2009¹⁷⁵ and followed by numerous studies performed to study the link between brain function and behavior using optogenetic stimulation.^176,177 None of the optogenetic studies allowing the delivery of treatment was performed in NPH. However, many applications of optogenetics in CNS diseases (stroke, epilepsy, multiple sclerosis, AD, and PD) were described by Ordaz et al.. One of the major obstacles to widely use optogenetic tools in patients remains the delivery to the brain. For this reason, it is essential to reduce brain tissue damage inflicted by probe penetrations and light-induced heating for optogenetic procedures. Once these problems are overcome, optogenetics can be an advantageous tool to treat neurological diseases providing an alternative treatment with less side effects than current therapies.¹⁷⁸ The first patient dosed with optogenetics was treated for retinitis pigmentosa in a clinical trial conducted by RetroSense Therapeutic in 2016. More recently, a phase I/II clinical trial of optogenetics for vision restoration is registered (NCT02556736, sponsor Allergan; NCT03326336, sponsor GenSight), but no results have been published to date.

HSC gene therapy to treat CNS diseases

Hematopoietic stem cell transplantation (HSCT) is the best example of stem-cell therapy and is currently an established treatment for several neurologically devastating inherited metabolic diseases, including ALD and lysosomal storage disorders (LSDs).^21,22 In LSDs, donor-derived microglia cells of myeloid origin are thought to be the source of enzyme after HSCT, cross-correcting the metabolic defect in affected host cells.¹⁷⁹ In addition, engrafted donor-derived cells may potentially help in reducing accumulated toxic substrates in the brain. Of importance, there is evidence that stem cells are not only replacing dying cells, but are also regulating inflammation and immune responses and have proneurogenic effects.¹⁸⁰

Numerous studies support the notion of using stem cells as a treatment for inherited diseases like LSDs,¹⁷⁹ HD, and also for complex diseases like AD, PD, and amyotrophic lateral sclerosis.^181,182 Further preclinical and clinical studies are needed to ensure the safety and efficacy of these treatment options.¹⁸²

Conclusion

The recent beneficial results demonstrated in phase I–II studies in human patients together with improved vector technology have placed gene therapy for CNS diseases in a new development paradigm. These approaches are no longer restricted to rare genetic diseases, but are being applied to common disease indications and pathways significantly expanding the scope of gene therapy for CNS indications. This expansion requires simplifying delivery protocols and anticipating the increasing need of vector production, particularly for IV targeting. Treating an increasing number of patients requires standardized delivery protocols suitable for adaptation in multiple centers around the world. Furthermore, manufacturability is a central component in expanding the GT field, not only the production of high quality and high quantity of vectors to meet future clinical demand, but also the cost and our capacity to make new gene therapy products accessible to all patients.

Footnotes

Author Disclosure

No competing financial interests exist.

Funding Information

No special fundings was received for this review.

References

Daneman

, Prat

. The blood–brain barrier. Cold Spring Harb Perspect Biol, 2015; 7:a020412.

Zlokovic

. The blood-brain barrier in health and chronic neurodegenerative disorders. Neuron, 2008; 57:178–201.

Hocquemiller

, Giersch

, Audrain

, et al. Adeno-associated virus-based gene therapy for CNS diseases. Hum Gene Ther, 2016; 27:478–496.

Tărlungeanu

, Novarino

. Genomics in neurodevelopmental disorders: an avenue to personalized medicine. Exp Mol Med, 2018; 50:100.

Gray

. Gene therapy and neurodevelopmental disorders. Neuropharmacology, 2013; 68:136–142.

Drew

. Gene therapy targets epilepsy. Nature, 2018; 564:S10–S11.

Kwiatkowska

, Nandhu

, Behera

, et al. Strategies in gene therapy for glioblastoma. Cancers (Basel), 2013; 5:1271–1305.

Guedon

J-MG

, Wu

, Zheng

, et al. Current gene therapy using viral vectors for chronic pain. Mol Pain, 2015; 11:27.

Tardieu

, Zérah

, Husson

, et al. Intracerebral administration of adeno-associated viral vector serotype rh.10 carrying human SGSH and SUMF1 cDNAs in children with mucopolysaccharidosis type IIIA disease: results of a phase I/II trial. Hum Gene Ther, 2014; 25:506–516.

10.

Tardieu

, Zérah

, Gougeon

M-L

, et al. Intracerebral gene therapy in children with mucopolysaccharidosis type IIIB syndrome: an uncontrolled phase 1/2 clinical trial. Lancet Neurol, 2017; 16:712–720.

11.

Zerah

, Piguet

, Colle

M-A

, 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.

12.

Mendell

, Al-Zaidy

, Shell

, et al. Single-dose gene-replacement therapy for spinal muscular atrophy. N Engl J Med, 2017; 377:1713–1722.

13.

Meyer

, Ferraiuolo

, Schmelzer

, et al. Improving single injection CSF delivery of AAV9-mediated gene therapy for SMA: a dose–response study in mice and nonhuman primates. Mol Ther, 2015; 23:477–487.

14.

Bestor

. Gene silencing as a threat to the success of gene therapy. J Clin Invest, 2000; 105:409–411.

15.

Rindt

, Yen

P-F

, Thebeau

, et al. Replacement of huntingtin exon 1 by trans-splicing. Cell Mol Life Sci, 2012; 69:4191–4204.

16.

Boussicault

, Alves

, Lamazière

, et al. CYP46A1, the rate-limiting enzyme for cholesterol degradation, is neuroprotective in Huntington's disease. Brain, 2016; 139:953–970.

17.

Burlot

M-A

, Braudeau

, Michaelsen-Preusse

, et al. Cholesterol 24-hydroxylase defect is implicated in memory impairments associated with Alzheimer-like Tau pathology. Hum Mol Genet, 2015; 24:5965–5976.

18.

Rose

, Dorard

, Audrain

, et al. Transient increase in sAPPα secretion in response to Aβ1–42 oligomers: an attempt of neuronal self-defense?. Neurobiol Aging, 2018; 61:23–35.

19.

Audrain

, Souchet

, Alves

, et al. βAPP processing drives gradual Tau pathology in an age-dependent amyloid rat model of Alzheimer's disease. Cereb Cortex, 2018; 28:3976–3993.

20.

Hulou

, Cho

, Chiocca

, et al. Experimental therapies: gene therapies and oncolytic viruses. Handbook of Clinical Neurology, 2016; 134:183–197.

21.

Sessa

, Lorioli

, Fumagalli

, et al. Lentiviral haemopoietic stem-cell gene therapy in early-onset metachromatic leukodystrophy: an ad-hoc analysis of a non-randomised, open-label, phase 1/2 trial. Lancet, 2016; 388:476–487.

22.

Eichler

, Duncan

, Musolino

, et al. Hematopoietic stem-cell gene therapy for cerebral adrenoleukodystrophy. N Engl J Med, 2017; 377:1630–1638.

23.

Piguet

, Alves

, Cartier

. Clinical gene therapy for neurodegenerative diseases: past, present, and future. Hum Gene Ther, 2017; 28:988–1003.

24.

Gao

, Vandenberghe

, Wilson

. New recombinant serotypes of AAV vectors. Curr Gene Ther, 2005; 5:285–297.

25.

Srivastava

, Carter

. AAV infection: protection from cancer. Hum Gene Ther, 2017; 28:323–327.

26.

Nault

, Mami

, La Bella

, et al. Wild-type AAV insertions in hepatocellular carcinoma do not inform debate over genotoxicity risk of vectorized AAV. Mol Ther, 2016; 24:660–661.

27.

Hastie

, Samulski

. Adeno-associated virus at 50: a golden anniversary of discovery, research, and gene therapy success—a personal perspective. Hum Gene Ther, 2015; 26:257–265.

28.

Ginn

, Amaya

, Alexander

, et al. Gene therapy clinical trials worldwide to 2017: an update. J Gene Med, 2018; 20:e3015.

29.

Pillay

, Carette

. Host determinants of adeno-associated viral vector entry. Curr Opin Virol, 2017; 24:124–131.

30.

Castle

, Turunen

, Vandenberghe

, Wolfe

. Controlling AAV tropism in the nervous system with natural and engineered capsids. In: Manfredsson F. (ed) Gene Therapy for Neurological Disorders. Methods in Molecular Biology. New York, NY: Humana Press,, 2016:133–149.

31.

Vandenberghe

, Wilson

, Gao

. Tailoring the AAV vector capsid for gene therapy. Gene Ther, 2009; 16:311–319.

32.

Auricchio

. Exchange of surface proteins impacts on viral vector cellular specificity and transduction characteristics: the retina as a model. Hum Mol Genet, 2001; 10:3075–3081.

33.

Hadaczek

, Stanek

, Ciesielska

, et al. Widespread AAV1- and AAV2-mediated transgene expression in the nonhuman primate brain: implications for Huntington's disease. Mol Ther Methods Clin Dev, 2016; 3:16037.

34.

Bartus

, Baumann

, Siffert

, et al. Safety/feasibility of targeting the substantia nigra with AAV2-neurturin in Parkinson patients. Neurology, 2013; 80:1698–1701.

35.

Chien

Y-H

, Lee

N-C

, Tseng

S-H

, et al. Efficacy and safety of AAV2 gene therapy in children with aromatic L-amino acid decarboxylase deficiency: an open-label, phase 1/2 trial. Lancet Child Adolesc Health, 2017; 1:265–273.

36.

Mittermeyer

, Christine

, Rosenbluth

, et al. Long-term evaluation of a phase 1 study of AADC gene therapy for Parkinson's disease. Hum Gene Ther, 2012; 23:377–381.

37.

Colle

M-A

, Piguet

, Bertrand

, et al. Efficient intracerebral delivery of AAV5 vector encoding human ARSA in non-human primate. Hum Mol Genet, 2010; 19:147–158.

38.

Foust

, Wang

, McGovern

, et al. Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN. Nat Biotechnol, 2010; 28:271–274.

39.

Murrey

, Naughton

, Duncan

, et al. Feasibility and safety of systemic rAAV9-h NAGLU delivery for treating mucopolysaccharidosis IIIB: toxicology, biodistribution, and immunological assessments in primates. Hum Gene Ther Clin Dev, 2014; 25:72–84.

40.

Lipinski

, Reid

, Boye

, et al. AAV vectors II 314. Systemic vascular transduction following intravenous injection of capsid mutant adeno-associated virus. Hum Gene Ther, 2015; 26:767–776.

41.

Gessler

, Tai

PWL

, Li

, Gao

. Intravenous infusion of AAV for widespread gene delivery to the nervous system. In: Castle M. (eds) Adeno-Associated Virus Vectors. Methods in Molecular Biology. New York, NY: Humana Press,, 2019:143–169.

42.

Deverman

, Pravdo

, Simpson

, et al. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat Biotechnol, 2016; 34:204–209.

43.

Hordeaux

, Wang

, Katz

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

44.

Zufferey

, Dull

, Mandel

, et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol, 1998; 72:9873–9880.

45.

Escors

, Breckpot

. Lentiviral vectors in gene therapy: their current status and future potential. Arch Immunol Ther Exp (Warsz), 2010; 58:107–119.

46.

Delzor

, Aurélie

, Escartin

, et al. Lentiviral vectors: a powerful tool to target astrocytes in vivo. Curr Drug Targets, 2013; 14:1336–1346.

47.

Hastie

, Cataldi

, Marriott

, et al. Understanding and altering cell tropism of vesicular stomatitis virus. Virus Res, 2013; 176:16–32.

48.

Carlotti

, Bazuine

, Kekarainen

, et al. Lentiviral vectors efficiently transduce quiescent mature 3T3–L1 adipocytes. Mol Ther, 2004; 9:209–217.

49.

Geng

, Doitsh

, Yang

, et al. Efficient delivery of lentiviral vectors into resting human CD4 T cells. Gene Ther, 2014; 21:444–449.

50.

Jakobsson

, Lundberg

. Lentiviral vectors for use in the central nervous system. Mol Ther, 2006; 13:484–493.

51.

Cartier

, Hacein-Bey-Abina

, Bartholomae

, et al. Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science, 2009; 326:818–823.

52.

Staal

FJT

, Aiuti

, Cavazzana

. Autologous stem-cell-based gene therapy for inherited disorders: state of the art and perspectives. Front Pediatr, 2019; 7:443.

53.

Stein

, Ott

, Schultze-Strasser

, et al. Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease. Nat Med, 2010; 16:198–204.

54.

Zychlinski

, Schambach

, Modlich

, et al. Physiological promoters reduce the genotoxic risk of integrating gene vectors. Mol Ther, 2008; 16:718–725.

55.

Poletti

, Charrier

, Corre

, et al. Preclinical development of a lentiviral vector for gene therapy of X-linked severe combined immunodeficiency. Mol Ther Methods Clin Dev, 2018; 9:257–269.

56.

Doi

, Takeuchi

. Gene therapy using retrovirus vectors: vector development and biosafety at clinical trials. Uirusu, 2015; 65:27–36.

57.

Rothe

, Modlich

, Schambach

. Biosafety challenges for use of lentiviral vectors in gene therapy. Curr Gene Ther, 2014; 13:453–468.

58.

Katsouri

, Lim

, Blondrath

, et al. PPARγ-coactivator-1α gene transfer reduces neuronal loss and amyloid-β generation by reducing β-secretase in an Alzheimer's disease model. Proc Natl Acad Sci U S A, 2016; 113:12292–12297.

59.

Palfi

, Gurruchaga

, Lepetit

, et al. Long-term follow-up of a phase I/II study of ProSavin, a lentiviral vector gene therapy for Parkinson's disease. Hum Gene Ther Clin Dev, 2018; 29:148–155.

60.

Azzouz

, Martin-Rendon

, Barber

, et al. Multicistronic lentiviral vector-mediated striatal gene transfer of aromatic L-amino acid decarboxylase, tyrosine hydroxylase, and GTP cyclohydrolase I induces sustained transgene expression, dopamine production, and functional improvement in a rat model of Parkinson's disease. J Neurosci, 2002; 22:10302–10312.

61.

Blagbrough

, Zara

. Animal models for target diseases in gene therapy—using DNA and siRNA delivery strategies. Pharm Res, 2009; 26:1–18.

62.

Hattori

, Sato

. Animal models of Parkinson's disease: similarities and differences between the disease and models. Neuropathology, 2007; 27:479–483.

63.

Lasbleiz

, Mestre-Francés

, Devau

, et al. Combining gene transfer and nonhuman primates to better understand and treat Parkinson's disease. Front Mol Neurosci, 2019; 12:10.

64.

Gopinath

, Nathar

, Ghosh

, et al. Contemporary animal models for human gene therapy applications. Curr Gene Ther, 2015; 15:531–540.

65.

Dawson

, Mandir

, Lee

. Animal models of PD: pieces of the same puzzle?. Neuron, 2002; 35:219–222.

66.

Bankiewicz

, Sudhakar

, Samaranch

, et al. AAV viral vector delivery to the brain by shape-conforming MR-guided infusions. J Control Release, 2016; 240:434–442.

67.

Samaranch

, Salegio

, San Sebastian

, et al. Strong cortical and spinal cord transduction after AAV7 and AAV9 delivery into the cerebrospinal fluid of nonhuman primates. Hum Gene Ther, 2013; 24:526–532.

68.

Salegio

, Samaranch

, Kells

, et al. Axonal transport of adeno-associated viral vectors is serotype-dependent. Gene Ther, 2013; 20:348–352.

69.

Kells

, Forsayeth

, Bankiewicz

. Glial-derived neurotrophic factor gene transfer for Parkinson's disease: anterograde distribution of AAV2 vectors in the primate brain. Neurobiol Dis, 2012; 48:228–235.

70.

Ciesielska

, Mittermeyer

, Hadaczek

, et al. Anterograde axonal transport of AAV2-GDNF in rat basal ganglia. Mol Ther, 2011; 19:922–927.

71.

Green

, Samaranch

, Zhang

, et al. Axonal transport of AAV9 in nonhuman primate brain. Gene Ther, 2016; 23:520–526.

72.

Evers

, Miniarikova

, Juhas

, et al. AAV5-miHTT gene therapy demonstrates broad distribution and strong human mutant Huntingtin lowering in a Huntington's disease minipig model. Mol Ther, 2018; 26:2163–2177.

73.

Hocquemiller

, Hemsley

, Douglass

, et al. AAVrh10 vector corrects disease pathology in MPS IIIA mice and achieves widespread distribution of SGSH in large animal brains. Mol Ther Methods Clin Dev, 2020; 17:174–187.

74.

Kordower

, Emborg

, Bloch

, et al. Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson's disease. Science (80-), 2000; 290:767–773.

75.

Jarraya

, Boulet

, Scott Ralph

, et al. Dopamine gene therapy for Parkinson's disease in a nonhuman primate without associated dyskinesia. Sci Transl Med, 2009; 1:2ra4.

76.

Linterman

, Palmer

, Kay

, et al. Lentiviral-mediated gene transfer to the sheep brain: implications for gene therapy in Batten disease. Hum Gene Ther, 2011; 22:1011–1020.

77.

Duque

, Arnold

, Odermatt

, et al. A large animal model of spinal muscular atrophy and correction of phenotype. Ann Neurol, 2015; 77:399–414.

78.

Katz

, Tecedor

, Chen

, et al. AAV gene transfer delays disease onset in a TPP1-deficient canine model of the late infantile form of Batten disease. Sci Transl Med, 2015; 7:313ra180.

79.

Samaranch

, Salegio

, San Sebastian

, et al. Adeno-associated virus serotype 9 transduction in the central nervous system of nonhuman primates. Hum Gene Ther, 2012; 23:382–389.

80.

Samaranch

, Sebastian

, Kells

, et al. AAV9-mediated expression of a non-self protein in nonhuman primate central nervous system triggers widespread neuroinflammation driven by antigen-presenting cell transduction. Mol Ther, 2014; 22:329–337.

81.

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.

82.

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.

83.

Passini

, Bu

, Richards

, et al. Translational fidelity of intrathecal delivery of self-complementary AAV9-survival motor neuron 1 for spinal muscular atrophy. Hum Gene Ther, 2014; 25:619–630.

84.

Hinderer

, Bell

, Vite

, et al. Widespread gene transfer in the central nervous system of cynomolgus macaques following delivery of AAV9 into the cisterna magna. Mol Ther Methods Clin Dev, 2014; 1:14051.

85.

Haurigot

, Marcó

, Ribera

, et al. Whole body correction of mucopolysaccharidosis IIIA by intracerebrospinal fluid gene therapy. J Clin Invest, 2013; 123:3254–3271.

86.

Gray

, Matagne

, Bachaboina

, et al. Preclinical differences of intravascular AAV9 delivery to neurons and glia: a comparative study of adult mice and nonhuman primates. Mol Ther, 2011; 19:1058–1069.

87.

Duque

, Joussemet

, Riviere

, et al. Intravenous administration of self-complementary AAV9 enables transgene delivery to adult motor neurons. Mol Ther, 2009; 17:1187–1196.

88.

Bucher

, Dubreil

, Colle

M-A

, et al. Intracisternal delivery of AAV9 results in oligodendrocyte and motor neuron transduction in the whole central nervous system of cats. Gene Ther, 2014; 21:522–528.

89.

Gurda

, De Guilhem De Lataillade

, Bell

, et al. Evaluation of AAV-mediated gene therapy for central nervous system disease in canine mucopolysaccharidosis VII. Mol Ther, 2016; 24:206–216.

90.

Hinderer

, Bell

, Louboutin

J-P

, et al. Neonatal systemic AAV induces tolerance to CNS gene therapy in MPS I dogs and nonhuman primates. Mol Ther, 2015; 23:1298–1307.

91.

Borel

, Gernoux

, Cardozo

, et al. Therapeutic rAAVrh10 mediated SOD1 silencing in adult SOD1 ^G93A mice and nonhuman primates. Hum Gene Ther, 2016; 27:19–31.

92.

Bevan

, Duque

, Foust

, et al. Systemic gene delivery in large species for targeting spinal cord, brain, and peripheral tissues for pediatric disorders. Mol Ther, 2011; 19:1971–1980.

93.

Yang

, Li

, Wang

, et al. Global CNS transduction of adult mice by intravenously delivered rAAVrh.8 and rAAVrh.10 and nonhuman primates by rAAVrh.10. Mol Ther, 2014; 22:1299–1309.

94.

Borel

, Gernoux

, Sun

, et al. Safe and effective superoxide dismutase 1 silencing using artificial microRNA in macaques. Sci Transl Med, 2018; 10:eaau6414.

95.

Hinderer

, Bell

, Katz

, et al. Evaluation of intrathecal routes of administration for adeno-associated viral vectors in large animals. Hum Gene Ther, 2018; 29:15–24.

96.

Bravo-Hernandez

, Tadokoro

, Navarro

, et al. Spinal subpial delivery of AAV9 enables widespread gene silencing and blocks motoneuron degeneration in ALS. Nat Med, 2020; 26:118–130.

97.

Tadokoro

, Miyanohara

, Navarro

, et al. Subpial adeno-associated virus 9 (AAV9) vector delivery in adult mice. J Vis Exp, 2017; 125:55770.

98.

Wolf

, Banerjee

, Hackett

, et al. Gene therapy for neurologic manifestations of mucopolysaccharidoses. Expert Opin Drug Deliv, 2015; 12:283–296.

99.

Wolfe

. Gene therapy in large animal models of human genetic diseases. Introduction. ILAR J, 2009; 50:107–111.

100.

Hordeaux

, Dubreil

, Robveille

, et al. Long-term neurologic and cardiac correction by intrathecal gene therapy in Pompe disease. Acta Neuropathol Commun, 2017; 5:66.

101.

Hordeaux

, Hinderer

, Goode

, et al. Toxicology study of intra-cisterna magna adeno-associated virus 9 expressing human alpha-L-iduronidase in rhesus macaques. Mol Ther Methods Clin Dev, 2018; 10:79–88.

102.

Kim

, Paek

, Nelson

, et al. Implementation of a chronic unilateral intraparenchymal drug delivery system in a swine model. J Neurosci Methods, 2014; 227:29–34.

103.

Brady

, Raghavan

, Alexander

, et al. Pathways of infusate loss during convection-enhanced delivery into the putamen nucleus. Stereotact Funct Neurosurg, 2013; 91:69–78.

104.

Belova

, Shaffer

, Trapa

. Insights from mathematical modeling for convection-enhanced intraputamenal delivery of GDNF. Med Biol Eng Comput, 2017; 55:2069–2077.

105.

Vazquez

, Hagel

, Willenberg

, et al. Polymer-coated cannulas for the reduction of backflow during intraparenchymal infusions. J Mater Sci Mater Med, 2012; 23:2037–2046.

106.

Ung

, Malone

, Canoll

, et al. Convection-enhanced delivery for glioblastoma: targeted delivery of antitumor therapeutics. CNS Oncol, 2015; 4:225–234.

107.

Lueshen

, Tangen

, Mehta

, et al. Backflow-free catheters for efficient and safe convection-enhanced delivery of therapeutics. Med Eng Phys, 2017; 45:15–24.

108.

Krauze

, Saito

, Noble

, et al. Reflux-free cannula for convection-enhanced high-speed delivery of therapeutic agents. J Neurosurg, 2005; 103:923–929.

109.

Lewis

, Woolley

, Johnson

, et al. Chronic, intermittent convection-enhanced delivery devices. J Neurosci Methods, 2016; 259:47–56.

110.

Debinski

, Tatter

. Convection-enhanced delivery for the treatment of brain tumors. Expert Rev Neurother, 2009; 9:1519–1527.

111.

Fan

, Nelson

, Ai

, et al. Continuous intraputamenal convection-enhanced delivery in adult rhesus macaques. J Neurosurg, 2015; 123:1569–1577.

112.

Bienemann

, White

, Woolley

, et al. The development of an implantable catheter system for chronic or intermittent convection-enhanced delivery. J Neurosci Methods, 2012; 203:284–291.

113.

Vogelbaum

, Brewer

, Barnett

, et al. First-in-human evaluation of the Cleveland Multiport Catheter for convection-enhanced delivery of topotecan in recurrent high-grade glioma: results of pilot trial 1. J Neurosurg, 2019; 130:476–485.

114.

Christine

, Bankiewicz

, Van Laar

, et al. Magnetic resonance imaging-guided phase 1 trial of putaminal AADC gene therapy for Parkinson's disease. Ann Neurol, 2019; 85:704–714.

115.

Nayak

, Herzog

. Progress and prospects: immune responses to viral vectors. Gene Ther, 2010; 17:295–304.

116.

Mingozzi

, Maus M

, Hui

, et al. CD8⁺ T-cell responses to adeno-associated virus capsid in humans. Nat Med, 2007; 13:419–422.

117.

Basner-Tschakarjan

, Mingozzi

. Cell-mediated immunity to AAV vectors, evolving concepts and potential solutions. Front Immunol, 2014; 5:350.

118.

Calcedo

, Wilson

. Humoral immune response to AAV. Front Immunol, 2013; 4:341.

119.

Rogers

, Martino

, Aslanidi

G V.

, et al. Innate immune responses to AAV vectors. Front Microbiol, 2011; 2:194.

120.

Calcedo

, Vandenberghe

, Gao

, et al. Worldwide epidemiology of neutralizing antibodies to adeno-associated viruses. J Infect Dis, 2009; 199:381–390.

121.

Veron

, Leborgne

, Monteilhet

, et al. Humoral and cellular capsid-specific immune responses to adeno-associated virus type 1 in randomized healthy donors. J Immunol, 2012; 188:6418–6424.

122.

Boutin

, Monteilhet

, Veron

, et al. Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors. Hum Gene Ther, 2010; 21:704–712.

123.

Wang

, Tapscott

, Chamberlain

, et al. Immunity and AAV-mediated gene therapy for muscular dystrophies in large animal models and human trials. Front Microbiol, 2011; 2:201.

124.

Banugaria

, Prater

, Ng

Y-K

, et al. The impact of antibodies on clinical outcomes in diseases treated with therapeutic protein: lessons learned from infantile Pompe disease. Genet Med, 2011; 13:729–736.

125.

Berrier

, Kazi

, Prater

, et al. CRIM-negative infantile Pompe disease: characterization of immune responses in patients treated with ERT monotherapy. Genet Med, 2015; 17:912–918.

126.

Ferreira

, Petry

, Salmon

. Immune responses to AAV-vectors, the glybera example from bench to bedside. Front Immunol, 2014; 5:82.

127.

Gaudet

, Méthot

, Déry

, et al. Efficacy and long-term safety of alipogene tiparvovec (AAV1-LPLS447X) gene therapy for lipoprotein lipase deficiency: an open-label trial. Gene Ther, 2013; 20:361–369.

128.

Manno

, Pierce

, Arruda

, et al. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med, 2006; 12:342–347.

129.

Marks

, Bartus

, Siffert

, et al. Gene delivery of AAV2-neurturin for Parkinson's disease: a double-blind, randomised, controlled trial. Lancet Neurol, 2010; 9:1164–1172.

130.

Christine

, Starr

, Larson

, et al. Safety and tolerability of putaminal AADC gene therapy for Parkinson disease. Neurology, 2009; 73:1662–1669.

131.

McPhee

SWJ

, Janson

, Li

, et al. Immune responses to AAV in a phase I study for Canavan disease. J Gene Med, 2006; 8:577–588.

132.

Kaplitt

, Feigin

, Tang

, et al. Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson's disease: an open label, phase I trial. Lancet, 2007; 369:2097–2105.

133.

Mingozzi

, Chen

, Edmonson

, et al. Prevalence and pharmacological modulation of humoral immunity to AAV vectors in gene transfer to synovial tissue. Gene Ther, 2013; 20:417–424.

134.

Corti

, Liberati

, Smith

, et al. Safety of intradiaphragmatic delivery of adeno-associated virus-mediated alpha-glucosidase (rAAV1-CMV-hGAA) gene therapy in children affected by Pompe disease. Hum Gene Ther Clin Dev, 2017; 28:208–218.

135.

Montenegro-Miranda

, ten Bloemendaal

, Kunne

, et al. Mycophenolate mofetil impairs transduction of single-stranded adeno-associated viral vectors. Hum Gene Ther, 2011; 22:605–612.

136.

Unzu

, Hervás-Stubbs

, Sampedro

, et al. Transient and intensive pharmacological immunosuppression fails to improve AAV-based liver gene transfer in non-human primates. J Transl Med, 2012; 10:122.

137.

Tseng

Y-S

, Agbandje-McKenna

. Mapping the AAV capsid host antibody response toward the development of second generation gene delivery vectors. Front Immunol, 2014; 5:9.

138.

Kishimoto

, Ferrari

, LaMothe

, et al. Improving the efficacy and safety of biologic drugs with tolerogenic nanoparticles. Nat Nanotechnol, 2016; 11:890–899.

139.

Meliani

, Boisgerault

, Hardet

, et al. Antigen-selective modulation of AAV immunogenicity with tolerogenic rapamycin nanoparticles enables successful vector re-administration. Nat Commun, 2018; 9:4098.

140.

Majowicz

, Maczuga

, Kwikkers

, et al. Mir-142-3p target sequences reduce transgene-directed immunogenicity following intramuscular adeno-associated virus 1 vector-mediated gene delivery. J Gene Med, 2013; 15:219–232.

141.

Annoni

, Brown

, Cantore

, et al. In vivo delivery of a microRNA-regulated transgene induces antigen-specific regulatory T cells and promotes immunologic tolerance. Blood, 2009; 114:5152–5161.

142.

Keeler

, ElMallah

, Flotte

. Gene therapy 2017: progress and future directions. Clin Transl Sci, 2017; 10:242–248.

143.

Chan

, Jang

, Yoo

, et al. Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Nat Neurosci, 2017; 20:1172–1179.

144.

Huang

, Chan

, Tobey

, et al. Delivering genes across the blood-brain barrier: LY6A, a novel cellular receptor for AAV-PHP.B capsids. bioRxiv, 2019; 538421.

145.

Hordeaux

, Yuan

, Clark

, et al. The GPI-linked protein LY6A drives AAV-PHP.B transport across the blood-brain barrier. Mol Ther, 2019; 27:912–921.

146.

Garg

, Bhandari

, Rath

, et al. Current strategies for targeted delivery of bio-active drug molecules in the treatment of brain tumor. J Drug Target, 2015; 23:865–887.

147.

Hynynen

, McDannold

, Vykhodtseva

, et al. Noninvasive MR imaging–guided focal opening of the blood-brain barrier in rabbits. Radiology, 2001; 220:640–646.

148.

Sheikov

, McDannold

, Vykhodtseva

, et al. Cellular mechanisms of the blood-brain barrier opening induced by ultrasound in presence of microbubbles. Ultrasound Med Biol, 2004; 30:979–989.

149.

Beccaria

, Canney

, Goldwirt

, et al. Opening of the blood-brain barrier with an unfocused ultrasound device in rabbits. J Neurosurg, 2013; 119:887–898.

150.

Hynynen

, McDannold

, Sheikov

, et al. Local and reversible blood–brain barrier disruption by noninvasive focused ultrasound at frequencies suitable for trans-skull sonications. Neuroimage, 2005; 24:12–20.

151.

Kobus

, Vykhodtseva

, Pilatou

, et al. Safety validation of repeated blood–brain barrier disruption using focused ultrasound. Ultrasound Med Biol, 2016; 42:481–492.

152.

McMahon

, Hynynen

. Acute inflammatory response following increased blood-brain barrier permeability induced by focused ultrasound is dependent on microbubble dose. Theranostics, 2017; 7:3989–4000.

153.

Marquet

, Tung

Y-S

, Teichert

, et al. Noninvasive, transient and selective blood-brain barrier opening in non-human primates in vivo. PLoS One, 2011; 6:e22598.

154.

Stavarache

, Petersen

, Jurgens

, et al. Safe and stable noninvasive focal gene delivery to the mammalian brain following focused ultrasound. J Neurosurg, 2019; 130:989–998.

155.

McDannold

, Clement

, Black

, et al. Transcranial magnetic resonance imaging- guided focused ultrasound surgery of brain tumors: initial findings in 3 patients. Neurosurgery, 2010; 66:323–332.

156.

Carpentier

, Canney

, Vignot

, et al. Clinical trial of blood-brain barrier disruption by pulsed ultrasound. Sci Transl Med, 2016; 8:343re2.

157.

Beccaria

, Canney

, Bouchoux

, et al. Blood-brain barrier disruption with low-intensity pulsed ultrasound for the treatment of pediatric brain tumors: a review and perspectives. Neurosurg Focus, 2020; 48:E10.

158.

Pouliopoulos

, Wu

, Burgess

, et al. A clinical system for non-invasive blood–brain barrier opening using a neuronavigation-guided single-element focused ultrasound transducer. Ultrasound Med Biol, 2020; 46:73–89.

159.

Ying

, Han

, Zeng

, et al. Evaluation of intervertebral disc regeneration with injection of mesenchymal stem cells encapsulated in PEGDA-microcryogel delivery system using quantitative T2 mapping: a study in canines. Am J Transl Res, 2019; 11:2028–2041.

160.

Fjord-Larsen

, Kusk

, Tornøe

, et al. Long-term delivery of nerve growth factor by encapsulated cell biodelivery in the Göttingen minipig basal forebrain. Mol Ther, 2010; 18:2164–2172.

161.

Emerich

, Winn

, Harper

, et al. Implants of polymer-encapsulated human NGF-secreting cells in the nonhuman primate: rescue and sprouting of degenerating cholinergic basal forebrain neurons. J Comp Neurol, 1994; 349:148–164.

162.

Zhang

, Hopkins

, Heier

, et al. Ciliary neurotrophic factor delivered by encapsulated cell intraocular implants for treatment of geographic atrophy in age-related macular degeneration. Proc Natl Acad Sci U S A, 2011; 108:6241–6245.

163.

Birch

, Bennett

, Duncan

, et al. Long-term follow-up of patients with retinitis pigmentosa receiving intraocular ciliary neurotrophic factor implants. Am J Ophthalmol, 2016; 170:10–14.

164.

Birch

, Weleber

, Duncan

, et al. Randomized trial of ciliary neurotrophic factor delivered by encapsulated cell intraocular implants for retinitis pigmentosa. Am J Ophthalmol, 2013; 156:283.e1–292.e1.

165.

Eriksdotter-Jönhagen

, Linderoth

, Lind

, et al. Encapsulated cell biodelivery of nerve growth factor to the basal forebrain in patients with Alzheimer's disease. Dement Geriatr Cogn Disord, 2012; 33:18–28.

166.

Bachoud-Lévi

A-C

, Déglon

, Nguyen

J-P

, et al. Neuroprotective gene therapy for Huntington's disease using a polymer encapsulated BHK cell line engineered to secrete human CNTF. Hum Gene Ther, 2000; 11:1723–1729.

167.

Bloch

, Bachoud-Lévi

, Déglon

, et al. Neuroprotective gene therapy for Huntington's disease, using polymer-encapsulated cells engineered to secrete human ciliary neurotrophic factor: results of a phase I study. Hum Gene Ther, 2004; 15:968–975.

168.

Aebischer

, Schluep

, Déglon

, et al. Intrathecal delivery of CNTF using encapsulated genetically modified xenogeneic cells in amyotrophic lateral sclerosis patients. Nat Med, 1996; 2:696–699.

169.

Lathuilière

, Schneider

. A bioactive implant to prevent Alzheimer disease. Med Sci, 2017; 33:81–84.

170.

Lathuilière

, Mach

, Schneider

. Encapsulated cellular implants for recombinant protein delivery and therapeutic modulation of the immune system. Int J Mol Sci, 2015; 16:10578–10600.

171.

Yizhar

, Fenno

, Davidson

, et al. Optogenetics in neural systems. Neuron, 2011; 71:9–34.

172.

Han

, Chow

, Zhou

, et al. A high-light sensitivity optical neural silencer: development and application to optogenetic control of non-human primate cortex. Front Syst Neurosci, 2011; 5:18.

173.

Galvan

, Stauffer

, Acker

, et al. Nonhuman primate optogenetics: recent advances and future directions. J Neurosci, 2017; 37:10894–10903.

174.

Ruiz

, Lustig

, Nassi

, et al. Optogenetics through windows on the brain in the nonhuman primate. J Neurophysiol, 2013; 110:1455–1467.

175.

Han

, Qian

, Bernstein

, et al. Millisecond-timescale optical control of neural dynamics in the nonhuman primate brain. Neuron, 2009; 62:191–198.

176.

Gerits

, Vanduffel

. Optogenetics in primates: a shining future?. Trends Genet, 2013; 29:403–411.

177.

Yazdan-Shahmorad

, Diaz-Botia

, Hanson

, et al. A large-scale interface for optogenetic stimulation and recording in nonhuman primates. Neuron, 2016; 89:927–939.

178.

Ordaz

, Wu

, Xu

X-M

. Optogenetics and its application in neural degeneration and regeneration. Neural Regen Res, 2017; 12:1197.

179.

Siddiqi

, Wolfe

. Stem cell therapy for the central nervous system in lysosomal storage diseases. Hum Gene Ther, 2016; 27:749–757.

180.

Sun

, Kurtzberg

. Cell therapy for diverse central nervous system disorders: inherited metabolic diseases and autism. Pediatr Res, 2018; 83:364–371.

181.

Ghosh

. Adult neurogenesis and the promise of adult neural stem cells. J Exp Neurosci, 2019; 13:117906951985687.

182.

Nguyen

, Zarriello

, Coats

, et al. Stem cell therapy for neurological disorders: a focus on aging. Neurobiol Dis, 2019; 126:85–104.

183.

Ellinwood

, Ausseil

, Desmaris

, et al. Safe, efficient, and reproducible gene therapy of the brain in the dog models of Sanfilippo and Hurler syndromes. Mol Ther, 2011; 19:251–259.

184.

Sondhi

, Peterson

, Giannaris

, et al. AAV2-mediated CLN2 gene transfer to rodent and non-human primate brain results in long-term TPP-I expression compatible with therapy for LINCL. Gene Ther, 2005; 12:1618–1632.

185.

Ciron

, Cressant

, Roux

, et al. Human α-iduronidase gene transfer mediated by adeno-associated virus types 1, 2, and 5 in the brain of nonhuman primates: vector diffusion and biodistribution. Hum Gene Ther, 2009; 20:350–360.

186.

Hadaczek

, Forsayeth

, Mirek

, et al. Transduction of nonhuman primate brain with adeno-associated virus serotype 1: vector trafficking and immune response. Hum Gene Ther, 2009; 20:225–237.

187.

Hadaczek

, Kohutnicka

, Krauze

, et al. Convection-enhanced delivery of adeno-associated virus type 2 (AAV2) into the striatum and transport of AAV2 within monkey brain. Hum Gene Ther, 2006; 17:291–302.

188.

Salegio

, Kells

, Richardson

, et al. Magnetic resonance imaging-guided delivery of adeno-associated virus type 2 to the primate brain for the treatment of lysosomal storage disorders. Hum Gene Ther, 2010; 21:1093–1103.

189.

Meneghini

, Lattanzi

, Tiradani

, et al. Pervasive supply of therapeutic lysosomal enzymes in the CNS of normal and Krabbe-affected non-human primates by intracerebral lentiviral gene therapy. EMBO Mol Med, 2016; 8:489–510.

190.

Naidoo

, Stanek

, Ohno

, et al. Extensive transduction and enhanced spread of a modified AAV2 capsid in the non-human primate CNS. Mol Ther, 2018; 26:2418–2430.

191.

, Ashe

, Bringas

, et al. Merits of combination cortical, subcortical, and cerebellar injections for thetreatment of Niemann-Pick disease type A. Mol Ther, 2012; 20:1893.

192.

Bucher

, Colle

M-A

, Wakeling

, et al. scAAV9 intracisternal delivery results in efficient gene transfer to the central nervous system of a feline model of motor neuron disease. Hum Gene Ther, 2013; 24:670–682.

193.

Ohno

, Samaranch

, Hadaczek

, et al. Kinetics and MR-based monitoring of AAV9 vector delivery into cerebrospinal fluid of nonhuman primates. Mol Ther Methods Clin Dev, 2019; 13:47–54.

194.

Gray

, Nagabhushan Kalburgi

, McCown

, et al. Global CNS gene delivery and evasion of anti-AAV-neutralizing antibodies by intrathecal AAV administration in non-human primates. Gene Ther, 2013; 20:450–459.

195.

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.

196.

Wang

, Yang

, Qiu

, et al. Widespread spinal cord transduction by intrathecal injection of rAAV delivers efficacious RNAi therapy for amyotrophic lateral sclerosis. Hum Mol Genet, 2014; 23:668–681.

197.

Choudhury

, Fitzpatrick

, Harris

, et al. In vivo selection yields AAV-B1 capsid for central nervous system and muscle gene therapy. Mol Ther, 2016; 24:1247–1257.

198.

LeWitt

, Rezai

, Leehey

, et al. AAV2-GAD gene therapy for advanced Parkinson's disease: a double-blind, sham-surgery controlled, randomised trial. Lancet Neurol, 2011; 10:309–319.

199.

Souweidane

, Fraser

, Arkin

, et al. Gene therapy for late infantile neuronal ceroid lipofuscinosis: neurosurgical considerations. J Neurosurg Pediatr, 2010; 6:115–122.

200.

Eberling

, Jagust

, Christine

, et al. Results from a phase I safety trial of hAADC gene therapy for Parkinson disease. Neurology, 2008; 70:1980–1983.

201.

Muramatsu

, Fujimoto

, Kato

, et al. A phase I study of aromatic L-amino acid decarboxylase gene therapy for Parkinson's disease. Mol Ther, 2010; 18:1731–1735.

202.

Lewis

, Woolley

, Johnson

, et al. Chronic, intermittent convection-enhanced delivery devices. J Neurosci Methods, 2016; 259:47–56.

203.

Miranpuri

, Hinchman

, Wang

, et al. Convection enhanced delivery: a comparison of infusion characteristics in ex vivo and in vivo non-human primate brain tissue. Ann Neurosci, 2013; 20:108–114.

204.

Brady

, Raghavan

, Singh

, et al. In vivo performance of a microfabricated catheter for intraparenchymal delivery. J Neurosci Methods, 2014; 229:76–83.

205.

Gill

, Barua

, Woolley

, et al. In vitro and in vivo testing of a novel recessed-step catheter for reflux-free convection-enhanced drug delivery to the brain. J Neurosci Methods, 2013; 219:1–9.

206.

Lewis

, Woolley

, Johnson

, et al. Maximising coverage of brain structures using controlled reflux, convection-enhanced delivery and the recessed step catheter. J Neurosci Methods, 2018; 308:337–345.

207.

Wernicke

, Lazow

, Taube

, et al. Surgical technique and clinically relevant resection cavity dynamics following implantation of cesium-131 brachytherapy in patients with brain metastases. Oper Neurosurg (Hagerstown), 2016; 12:49–60.

208.

Singleton

, Collins

, Bienemann

, et al. Convection enhanced delivery of panobinostat (LBH589)-loaded pluronic nano-micelles prolongs survival in the F98 rat glioma model. Int J Nanomedicine, 2017; 12:1385–1399.

The Challenge of Gene Therapy for Neurological Diseases: Strategies and Tools to Achieve Efficient Delivery to the Central Nervous System

Abstract

Introduction

Evaluation of Administration Routes in Preclinical Studies

Intraparenchymal delivery

Intracerebrospinal fluid delivery

Intravenous delivery

Routes of Administration in Clinical Studies

Intracerebrospinal fluid delivery

Intrathecal lumbar administration with two potential modalities

Intracisternal administration 100,101

Intraventricular administration

Intraparenchymal delivery

Technical Aspects: State of the Art

Devices for intraparenchymal delivery of therapeutics agents

Principles

Catheters subtypes

Design

Pumps and syringes

Risks Assessment

Linked to the delivery procedure

Linked to the treatment

Future Development and Perspectives

New AAV serotypes with broad CNS transduction after IV delivery

BBB opening as a solution to enhance CNS targeting

Encapsulated cells and optogenetics

HSC gene therapy to treat CNS diseases

Conclusion

Footnotes

Author Disclosure

Funding Information

References

Intracisternal administration^100,101