Central Nervous System Gene Transfer Research Articles

Safety Study of AAV hTert and Klotho Gene Transfer Therapy for Dementia

A sponsored, interventional, non-randomized study without a control group using a novel and proprietary central nervous system gene transfer method to deliver AAV hTert and Klotho genes to five patients with mild or moderate dementia was performed to primarily evaluate safety. Clinical response data was gathered as a secondary interest. The therapy demonstrated a very high safety profile with no serious adverse effects identified. Clinical evaluation of the patients over the course of the one year follow up yielded significant findings with all five patients demonstrating evident reversal of Dementia symptoms such as sustained cognitive improvement as measured by the Folstein exam. Telomere analysis was performed before and after the therapy. A measurable elongation of the participants telomeres was identified, and biological age was reduced as chronological age increased.

Widespread Central Nervous System Gene Transfer and Silencing After Systemic Delivery of Novel AAV-AS Vector

Effective gene delivery to the central nervous system (CNS) is vital for development of novel gene therapies for neurological diseases. Adeno-associated virus (AAV) vectors have emerged as an effective platform for in vivo gene transfer, but overall neuronal transduction efficiency of vectors derived from naturally occurring AAV capsids after systemic administration is relatively low. Here, we investigated the possibility of improving CNS transduction of existing AAV capsids by genetically fusing peptides to the N-terminus of VP2 capsid protein. A novel vector AAV-AS, generated by the insertion of a poly-alanine peptide, is capable of extensive gene transfer throughout the CNS after systemic administration in adult mice. AAV-AS is 6- and 15-fold more efficient than AAV9 in spinal cord and cerebrum, respectively. The neuronal transduction profile varies across brain regions but is particularly high in the striatum where AAV-AS transduces 36% of striatal neurons. Widespread neuronal gene transfer was also documented in cat brain and spinal cord. A single intravenous injection of an AAV-AS vector encoding an artificial microRNA targeting huntingtin (Htt) resulted in 33-50% knockdown of Htt across multiple CNS structures in adult mice. This novel AAV-AS vector is a promising platform to develop new gene therapies for neurodegenerative disorders.

388. Wide-Scale Gene Transfer of Amyotrophic Lateral Sclerosis Related Proteins To Adult Rats

Wide-scale gene transfer to the central nervous system (CNS) has been achieved in the past few years by way of a peripheral route of administration. This may be relevant to gene therapy considering that a one-time peripheral treatment can penetrate the CNS and affect CNS function long-term. We have used the wide-scale approach to study a disease of the spinal cord, amyotrophic lateral sclerosis (ALS). TDP-43 and FUS are RNA-binding proteins involved in the genetics and pathology of ALS and frontotemporal lobar degeneration (FTLD). Most cases of ALS, including sporadic, non-familial ALS, harbor TDP-43 pathology. TDP-43 was previously expressed in the spinal cord among other tissues after an intravenous delivery of AAV9 to neonatal rats. An adult onset model would be more relevant to ALS and more amenable for sequential gene therapy or pharmacological interventions. In this study, we attempted to induce motor paralysis relevant to ALS in adult rats by expressing TDP-43 or FUS. Several AAV vectors encoding GFP were tested for optimization, AAV8, 9, and 10. The vectors were applied via tail vein to young rats (6 weeks old) at an equal per kg vector dose. Gene transfer in the adult rat CNS was feasible and highly neuron-selective as observed in the spinal cord and the cerebellum with all AAVs tested. When TDP-43 or FUS were individually expressed in adults with AAV9, both proteins caused severe immobilization within 4 weeks, and in the case of FUS, there was physical atrophy. With refinement, this approach could yield an appropriate platform for studying the mechanisms and molecular therapy of ALS and FTLD.

Expanded Central Nervous System Gene Transfer in Rats by Intravenous Delivery of Recombinant Adeno-Associated Virus

Viral vector gene transfer is an important tool for studying protein function in vivo. Recombinant adeno-associated virus (AAV) is advantageous for gene transfer to the central nervous system. Over the past few years it has been shown that an intravenous vector delivery can transduce neurons throughout the central nervous system [1-3]. The images are of rats administered AAV serotype 9 vector encoding green fluorescent protein (GFP). The rats were injected intravenously on post-natal day one and imaged for GFP expression 4-12 weeks later. There is robust expression in the spinal cord and dorsal root ganglia and the cerebellum (Figures 1-3). We have been exploiting the efficient spinal cord transduction to study a spinal cord disease, amyotrophic lateral sclerosis [2-4].

Global CNS gene delivery and evasion of anti-AAV-neutralizing antibodies by intrathecal AAV administration in non-human primates

Injection of AAV into the cerebrospinal fluid (CSF) offers a means to achieve widespread transgene delivery to the central nervous system, where the doses can be readily translated from small to large animals. In contrast to studies with other serotypes (AAV2, AAV4, AAV5) in rodents, we report that a naturally-occurring capsid (AAV9) and rationally-engineered capsid (AAV2.5) are able to achieve broad transduction throughout the brain and spinal cord parenchyma following a single injection into the CSF (via cisterna magna or lumbar cistern) in non-human primates (NHP). Using either vector at a dose of ~2×1012 vg per 3-6 kg animal, approximately 2% of the entire brain and spinal cord was transduced, covering all regions of the CNS. AAV9 in particular displayed efficient transduction of spinal cord motor neurons. The peripheral organ biodistribution was highly reduced compared to intravascular delivery, and the presence of circulating anti-AAV neutralizing antibodies up to a 1:128 titer had no inhibitory effect on CNS gene transfer. Intra-CSF delivery effectively translates from rodents to NHPs, which provides encouragement for the use of this approach in humans to treat motor neuron and lysosomal storage diseases.

Targeted gene delivery to the mouse brain by MRI-guided focused ultrasound-induced blood–brain barrier disruption

This study aimed to investigate the feasibility of targeted gene transfer into central nervous system (CNS) by MRI-guided focused ultrasound-induced blood–brain barrier (BBB) disruption. Before each sonication, T2-weighted images were obtained to select the target region. Followed by injecting DNA-loaded microbubbles into the tail vein, sonication was performed. The state of local BBB, distribution of plasmid DNA through the opened BBB, the ultrastructural changes of neurons and BDNF expression were detected. The results showed that MRI-guided focused ultrasound (FUS) could accomplish noninvasive, transient, and local BBB disruption, at 1h after sonication, plasmid DNA across the opened BBB had been internalized into the neurons presenting heterogeneous distribution and numerous transparent vesicles were observed in the cytoplasm of the neurons at the sonicated region, suggesting vesicle-mediated endocytosis. At 48h after sonication, the expressions of exogenous gene pBDNF-EGFP were observed in the cytoplasm of some neurons, and BDNF expressions were markedly enhanced by the combination of ultrasound and pBDNF-EGFP-loaded microbubbles about 20-fold than that of the control group (P<0.01). The method by using MRI-guided FUS to induce the local BBB disruption could accomplish effective targeted exogenous gene transfer in CNS. This technique may provide a new option for the treatment of various CNS diseases.

Several rAAV Vectors Efficiently Cross the Blood–brain Barrier and Transduce Neurons and Astrocytes in the Neonatal Mouse Central Nervous System

Noninvasive systemic gene delivery to the central nervous system (CNS) has largely been impeded by the blood-brain barrier (BBB). Recent studies documented widespread CNS gene transfer after intravascular delivery of recombinant adeno-associated virus 9 (rAAV9). To investigate alternative and possibly more potent rAAV vectors for systemic gene delivery across the BBB, we systematically evaluated the CNS gene transfer properties of nine different rAAVEGFP vectors after intravascular infusion in neonatal mice. Several rAAVs efficiently transduce neurons, motor neurons, astrocytes, and Purkinje cells; among them, rAAVrh.10 is at least as efficient as rAAV9 in many of the regions examined. Importantly, intravenously delivered rAAVs did not cause abnormal microgliosis in the CNS. The rAAVs that achieve stable widespread gene transfer in the CNS are exceptionally useful platforms for the development of therapeutic approaches for neurological disorders affecting large regions of the CNS as well as convenient biological tools for neuroscience research.

Expansive Gene Transfer in the Rat CNS Rapidly Produces Amyotrophic Lateral Sclerosis Relevant Sequelae When TDP-43 is Overexpressed

Improved spread of transduction in the central nervous system (CNS) was achieved from intravenous administration of adeno-associated virus serotype-9 (AAV9) to neonatal rats. Spinal lower motor neuron transduction efficiency was estimated to be 78% using the highest vector dose tested at a 12-week interval. The widespread expression could aid studying diseases that affect both the spinal cord and brain, such as amyotrophic lateral sclerosis (ALS). The protein most relevant to neuropathology in ALS is transactive response DNA-binding protein 43 (TDP-43). When expressed in rats, human wild-type TDP-43 rapidly produced symptoms germane to ALS including paralysis of the hindlimbs and muscle wasting, and mortality over 4 weeks that did not occur in controls. The hindlimb atrophy and weakness was evidenced by assessments of rotarod, rearing, overall locomotion, muscle mass, and histology. The muscle wasting suggested denervation, but there was only 14% loss of motor neurons in the TDP-43 rats. Tissues were negative for ubiquitinated, cytoplasmic TDP-43 pathology, suggesting that altering TDP-43's nuclear function was sufficient to cause the disease state. Other relevant pathology in the rats included microgliosis and degenerating neuronal perikarya positive for phospho-neurofilament. The expression pattern encompassed the distribution of neuropathology of ALS, and could provide a rapid, relevant screening assay for TDP-43 variants and other disease-related proteins.

896. Long Term Transgene Expression in the Brain Delivered by SV40-Derived Vectors

Top of pageAbstract Gene transfer to the central nervous system (CNS) has been studied using various vectors. Recombinant, Tag-deleted SV40 vectors provide long-term transgene expression in vitro and in vivo in multiple non-CNS cell types, and can be used in vitro to transduce both NT2-derived and primary human neurons, and primary human brain microglia. We previously reported administering a rSV40 vector into the caudate-putamen (CP) of the rat brain using stereotaxic conditions. This vector carries HIV-1 Nef protein with a carboxyl-terminal FLAG epitope tag driven with a CMV-IEP promoter, SV(Nef-FLAG). Transgene expression was assessed 4, 7 or 14 days later by immunostaining of serial coronal cryostat sections. Numerous transgene-positive cells were seen at those times, at least several mm longitudinally away from the injection site. The present study aimed to determine whether these vectors could also deliver long term expression of a transgene, up to 3 months after unilateral injection into the CP, hippocampus, and cerebellum (lobe 6). The uninjected side, or saline injection, were used as controls. No cells on the uninjected side were immunopositive for FLAG.Sections incubated with non-immune primary antibody (immunostain negative controls) were also negative. The number of transgene-positive cells counted 1, 2 and 3 months after injection of the vector in the CP was higher than the one at d14, and showed a wider anatomic distribution. Transgene-positive cells were seen not only in the CP, but also in different brain structures along the needle track. The longitudinal extent of the transduced area was similar along the whole CP for time points greater than 4 days. In the CP, many transgene-expressing cells stained positively for NeuN, or MAP2, indicating a substantial neuronal transduction, with more than 95% of the transduced cells co-immunostaining with these neuronal markers. Astrocytes generally did not express the transgene. Long term transgene expression was also seen in the hippocampus (dentate gyrus) and in the cerebellum. More transgene-positive cells were observed when 10 microl of vector were given than with an injection of 1 microl. In the cerebellum, transgene-positive cells colocalized with calbindin, a marker for Purkinje cells. Previous studies of rSV40 gene delivery did not identify inflammatory or immunologic responses to the vector. In these studies, we also looked for evidence of inflammatory or immune reaction to the transduction process, using sections stained with neutral red (NR): no inflammation was seen at any time point in brains injected in any of the different structures. Likewise, no CD8-positive T cells were identified in sections of brains injected with SV(Nef-FLAG). These results show that SV40-derived vectors can be used to achieve long term and efficient CNS gene transfer without evidence of inflammatory or immune reaction, preferentially to neurons and to a lesser extent, microglial cells, when injected in different parenchymal structures of the rat brain.

AAV2-mediated CLN2 gene transfer to rodent and non-human primate brain results in long-term TPP-I expression compatible with therapy for LINCL

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal, autosomal recessive disease resulting from mutations in the CLN2 gene with consequent deficiency in its product tripeptidyl peptidase I (TPP-I). In the central nervous system (CNS), the deficiency of TPP-I results in the accumulation of proteins in lysosomes leading to a loss of neurons causing progressive neurological decline, and death by ages 10-12 years. To establish the feasibility of treating the CNS manifestations of LINCL by gene transfer, an adeno-associated virus 2 (AAV2) vector encoding the human CLN2 cDNA (AAV2CUhCLN2) was assessed for its ability to establish therapeutic levels of TPP-I in the brain. In vitro studies demonstrated that AAV2CUhCLN2 expressed CLN2 and produced biologically active TPP-I protein of which a fraction was secreted as the pro-TPP-I precursor and was taken up by nontransduced cells (ie, cross-correction). Following AAV2-mediated CLN2 delivery to the rat striatum, enzymatically active TPP-I protein was detected. By immunohistochemistry TPP-I protein was detected in striatal neurons (encompassing nearly half of the target structure) for up to 18 months. At the longer time points following striatal administration, TPP-I-positive cell bodies were also observed in the substantia nigra, frontal cerebral cortex and thalamus of the injected hemisphere, and the frontal cerebral cortex of the noninjected hemisphere. These areas of the brain contain neurons that extend axons into the striatum, suggesting that CNS circuitry may aid the distribution of the gene product. To assess the feasibility of human CNS delivery, a total of 3.6 x 10(11) particle units of AAV2CUhCLN2 was administered to the CNS of African green monkeys in 12 distributed doses. 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. Together, these data support the development of direct CNS gene transfer using an AAV2 vector expressing the CLN2 cDNA for the CNS manifestations of LINCL.

1063. In Utero β-Glucuronidase Gene Transfer with AAV Vectors Results in Widespread Transduction, Enzyme Activity, and Disease Correction in the MPS VII Mouse Brain

Mucopolysaccharidosis (MPS) VII is a heritable lysosomal storage disease caused by the deficiency of b-glucuronidase (GUSB) enzyme. MPS VII is a chronic and progressive multiorgan disorder with signs of pathology already present during fetal life. Most therapeutic approaches for MPS VII have focused on postnatal treatments. Although these approaches can reverse storage lesions, the central nervous system (CNS) is more difficult to treat. We investigated in utero gene transfer in the MPS VII mouse CNS with AAV 1 and AAV2. We injected 1 μl of AAV1 (3.3×1013 genomes/ml) or AAV2 (1.4×1012 genomes/ml) into the lateral ventricle of the fetal brain on day 15.5 of gestation. AAV1 injected brains demonstrated extensive and widespread GUSB activity and mRNA expression the brain. There was retrograde transport of GUSB to the ganglion cells of the retina. GUSB activity was detected in the gray matter throughout most of the spinal cord segments. Storage lesions were reversed in the brain and spinal cord. Ubiquitin immunoreactivity was significantly reduced compared to the affected control. This suggested that in utero gene transfer with AAV1 corrected the disease and prevented the subsequent neurodegeneration. The AAV2 treated brains were treated with a lower dose of vector than the AAV1 experimental group, and these brains have significantly lower levels of enzyme and vector transduction. Very few GUSB positive ganglion cells were detected in the retina, and low levels of GUSB activity was present in the gray matter of the spinal cord. Evaluation of disease correction in the eye, spinal cord and brain for the AAV2 treated brains is ongoing. These results suggest that fetal gene therapy with AAV vectors may provide a therapeutic strategy capable widespread enzyme distribution and disease correction in the CNS.

CNS gene transfer mediated by a novel controlled release system based on DNA complexes of degradable polycation PPE-EA: a comparison with polyethylenimine/DNA complexes

Nonviral gene delivery systems based upon polycation/plasmid DNA complexes are quickly gaining recognition as an alternative to viral gene vectors for their potential in avoiding immunogenicity and toxicity problems inherent in viral systems. We investigated in this study the feasibility of using a controlled release system based on DNA complexed with a recently developed polymeric gene carrier, polyaminoethyl propylene phosphate (PPE-EA), to achieve gene transfer in the brain. A unique feature of this gene delivery system is the biodegradability of PPE-EA, which can provide a sustained release of DNA at different rates depending on the charge ratio of the polymer to DNA. PPE-EA/DNA complexes, naked DNA, and DNA complexed with polyethylenimine (PEI), a nondegradable cationic polymer known to be an effective gene carrier, were injected intracisternally into the mouse cerebrospinal fluid. Transgene expression mediated by naked DNA was mainly detected in the brain stem, a region close to the injection site. With either PPE-EA or PEI as a carrier, higher levels of gene expression could be detected in the cerebral cortex, basal ganglia, and diencephalons. Transgene expression in the brain mediated by PPE-EA/DNA complexes at an N/P ratio of 2 persisted for at least 4 weeks, with a significant higher level than that produced by either naked plasmid DNA or PEI/DNA at the 4-week time point. Furthermore, PPE-EA displayed much lower toxicity in cultured neural cells as compared to PEI and did not cause detectable pathological changes in the central nervous system (CNS). The results established the potential of PPE-EA as a new and biocompatible gene carrier to achieve sustained gene expression in the CNS.

From the Cover: Recombinant adeno-associated virus type 2, 4, and 5 vectors: Transduction of variant cell types and regions in the mammalian central nervous system

Recombinant adeno-associated virus vectors based on serotype 2 (rAAV2) can direct transgene expression in the central nervous system (CNS), but it is not known how other rAAV serotypes perform as CNS gene transfer vectors. Serotypes 4 and 5 are distinct from rAAV2 and from each other in their capsid regions, suggesting that they may direct binding and entry into different cell types. In this study, we examined the tropisms and transduction efficiencies of beta-galactosidase-encoding vectors made from rAAV4 and rAAV5 compared with similarly designed rAAV2-based vectors. Injection of rAAV5 beta-galactosidase (betagal) or rAAV4betagal into the lateral ventricle resulted in stable transduction of ependymal cells, with approximately 10-fold more positive cells than in mice injected with rAAV2betagal. Major differences between the three vectors were revealed upon striatal injections. Intrastriatal injection of rAAV4betagal resulted again in striking ependyma-specific expression of transgene, with a notable absence of transduced cells in the parenchyma. rAAV2betagal and rAAV5betagal intrastriatal injections led to beta-gal-positive parenchymal cells, but, unlike rAAV2betagal, rAAV5betagal transduced both neurons and astrocytes. The number of transgene-positive cells in rAAV5betagal-injected brains was 130 and 5,000 times higher than in rAAV2betagal-injected brains at 3 and 15 wk, respectively. Moreover, transgene-positive cells were widely dispersed throughout the injected hemisphere in rAAV5betagal-transduced animals. Together, our data provide in vivo support for earlier in vitro work, suggesting that rAAV4 and rAAV5 gain cell entry by means of receptors distinct from rAAV2. These differences could be exploited to improve gene therapy for CNS disorders.

Adeno-associated virus-mediated gene transfer to the brain: duration and modulation of expression.

Adeno-associated virus (AAV) is a promising vector for central nervous system (CNS) gene transfer, but a number of issues must be addressed if AAV is to be used for widespread delivery throughout the CNS. Our aim was to test the effect of dose, route of delivery, and hydroxyurea treatment on brain expression of beta-galactosidase activity after cerebral inoculation with an rAAV-lacZ vector (rAAV-beta-gal). We also wished to test whether an immune response appeared against the vector and the transgene product. We found in BALB/c mice that beta-Gal expression increased during the first 2 months after inoculation, then decreased slightly by 4 months, and continued out to 6, 12, and 15 months in single animals. Cerebral injection produced localized beta-Gal expression that did not diffuse to other regions despite a fivefold increase in injection volume. Intraventricular injection resulted in negligible transduction. Antibodies to AAV capsid protein and beta-Gal appeared at low levels at 2 and 4 months, but correlated poorly with beta-Gal expression and did not prevent readministration of rAAV-beta-gal. Hydroxyurea treatment did not result in increased transduction in vivo. We conclude that our study confirms rAAV vectors as having considerable potential for CNS gene transfer; however, several important problems must be addressed if this vector system is to be used for long-term transduction of the entire brain. Sustained, regulatable expression will be needed if rAAV is to be used in the treatment of chronic CNS disease. The difficulty in delivering AAV to diverse regions of the brain is an important problem that must be overcome if these vectors are to be used for anything beyond localized transduction.

Lipofectin-facilitated transfer of cholecystokinin gene corrects behavioral abnormalities of rats with audiogenic seizures

To evaluate the potential for lipofectin-mediated central nervous system gene transfer, the plasmid coding for cholecystokinin was administered intracerebroventricularly to rats, which have congenital audiogenic seizures and high responses to peripheral electric stimulation-induced analgesia. Previous studies had shown that low brain cholecystokinin levels may be the neurochemical variable of rat's audiogenic seizure and high responses to the analgesia because cholecystokinin is an anticonvulsant and anti-opioid neuropeptide. Gene transfer of cholecystokinin corrected the increased susceptibility to audiogenic seizures and the high responses to analgesia for about one week. Similar administration of plasmid expressing β-galactosidase indicated that the vector mainly transfected ependymal cells lining the ventricle and pia mater cells. The increased cholecystokinin messenger RNA and immunoreactivity in the hippocampus following stereotactic intrahippocampal administration of cholecystokinin plasmid was also demonstrated with in situ hybridization and immunohistochemistry techniques. These results suggest that lipofectin-mediated gene transfer will be useful for studies of brain function, the modification of behavior and gene therapy for central nervous system disorders.