In-vivo Gene Therapy Research Articles

Drug-induced liver injury related to gene therapy: A new challenge to be managed.

Gene therapy is being successfully developed for the treatment of several genetic disorders. Various methods of gene transfer have been developed to enable the production of the deficient enzyme or protein. One of the most important is adeno-associated virus vectors, which have been shown to be viable for use in invivo gene therapy. Several gene therapies have already been approved. They are also promising for acquired diseases. Important examples include gene therapy for haemophilia A and B, X-linked myotubular myopathy, spinal muscular atrophy and several liver diseases such as Criggler-Najjar disease, alpha-1 antitrypsin deficiency and Fabry disease. However, the introduction of a foreign compound into hepatocytes leads to hepatic reactions with heterogeneous phenotypic expression and a wide spectrum of severity, ranging from mild transaminase elevation to acute liver failure. Several mechanisms appear to be involved in liver injury, including an immune response, but also direct toxicity depending on the method of gene transfer. As a result, the incidence, expression and severity of liver injury vary from indication to indication and from patient to patient. Patients treated for haemophilia A are more prone to transaminase elevation than those treated for haemophilia B. Corticosteroids are successfully used to correct liver reactions but also to prevent degradation of the transferred gene and loss of therapeutic activity. The aim of this review is to describe the risk of liver injury according to the indication for gene therapy and the short- and long-term management currently proposed to prevent or correct liver reactions in clinical practice.

Scalable inference of cell differentiation networks in gene therapy clonal tracking studies of haematopoiesis.

Investigating cell differentiation under a genetic disorder offers the potential for improving current gene therapy strategies. Clonal tracking provides a basis for mathematical modelling of population stem cell dynamics that sustain the blood cell formation, a process known as haematopoiesis. However, many clonal tracking protocols rely on a subset of cell types for the characterization of the stem cell output, and the data generated are subject to measurement errors and noise. We propose a stochastic framework to infer dynamic models of cell differentiation from clonal tracking data. A state-space formulation combines a stochastic quasi-reaction network, describing cell differentiation, with a Gaussian measurement model accounting for data errors and noise. We developed an inference algorithm based on an extended Kalman filter, a nonlinear optimization, and a Rauch-Tung-Striebelsmoother. Simulations show that our proposed method outperforms the state-of-the-art and scales to complex structures of cell differentiations in terms of nodes size and network depth. The application of our method to five in vivo gene therapy studies reveals different dynamics of cell differentiation. Our tool can provide statistical support to biologists and clinicians to better understand cell differentiation and haematopoietic reconstitution after a gene therapy treatment. The equations of the state-space model can be modified to infer other dynamics besides cell differentiation. The stochastic framework is implemented in the R package Karen which is available for download at https://cran.r-project.org/package=Karen. The code that supports the findings of this study is openly available at https://github.com/delcore-luca/CellDifferentiationNetworks.

An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis

SpCas9 and AsCas12a are widely utilized as genome-editing tools in human cells. However, their relatively large size poses a limitation for delivery by cargo-size-limited adeno-associated virus (AAV) vectors. The type V-F Cas12f from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been harnessed as a compact genome-editing tool. Here, we developed an approach, combining deep mutational scanning and structure-informed design, to successfully generate two AsCas12f activity-enhanced (enAsCas12f) variants. Remarkably, the enAsCas12f variants exhibited genome-editing activities in human cells comparable with those of SpCas9 and AsCas12a. The cryoelectron microscopy (cryo-EM) structures revealed that the mutations stabilize the dimer formation and reinforce interactions with nucleic acids to enhance their DNA cleavage activities. Moreover, enAsCas12f packaged with partner genes in an all-in-one AAV vector exhibited efficient knock-in/knock-out activities and transcriptional activation in mice. Taken together, enAsCas12f variants could offer a minimal genome-editing platform for invivo gene therapy.

Unveiling the secrets of adeno-associated virus: novel high-throughput approaches for the quantification of multiple serotypes

Adeno-associated virus (AAV) vectors are among the most prominent viral vectors for invivo gene therapy, and their investigation and development using high-throughput techniques have gained increasing interest. However, sample throughput remains a bottleneck in most analytical assays. In this study, we compared commonly used analytical methods for AAV genome titer, capsid titer, and transducing titer determination with advanced methods using AAV2, AAV5, and AAV8 as representative examples. For the determination of genomic titers, we evaluated the suitability of qPCR and four different digital PCR methods and assessed the respective advantages and limitations of each method. We found that both ELISA and bio-layer interferometry provide comparable capsid titers, with bio-layer interferometry reducing the workload and having a 2.8-fold higher linear measurement range. Determination of the transducing titer demonstrated that live-cell analysis required less manual effort compared with flow cytometry. Both techniques had a similar linear range of detection, and no statistically significant differences in transducing titers were observed. This study demonstrated that the use of advanced analytical methods provides faster and more robust results while simultaneously increasing sample throughput and reducing active bench work time.

Recent advances in upstream process development for production of recombinant adeno-associated virus.

Recombinant adeno-associated virus (rAAV) is rapidly emerging as the preferred delivery vehicle for gene therapies, with promising advantages in safety and efficacy. Key challenges in systemic in-vivo rAAV gene therapy applications are the gap in production capabilities versus potential market demand and complex production process. This review summarizes currentavailable information on rAAV upstream manufacturing processes and proposed optimizations for production. The advancements in rAAV production media were reviewed with proposals to speed up the cell culture process development. Furthermore, major methods for genetic element delivery to host cells were summarized with their advantages, limitations, and future directions for optimization. In addition, culture vessel selection criteria were listed based on production cell system, scale, and development stage. Process control at the production step was also outlined with an in-depth understanding of production kinetics and quality control.

Capsid-modified adeno-associated virus vectors as novel vaccine platform for cancer immunotherapy

Immunotherapy has significantly improved treatment outcomes in various cancer entities. To enhance immunogenicity and efficacy, and to further broaden its applicability, co-administration of anti-tumor vaccines is considered as a promising strategy. Here, we introduce adeno-associated virus (AAV) vectors, widely used for invivo gene therapy, as a potent cancer vaccine platform. Our AAV vector-based vaccine combines antigen display on the capsid surface with a vector-mediated antigen overexpression targeting different components of the immune system in a unique chronological order by a single intramuscular application. Thereby, both profound and long-lasting antigen-specific T and B cell immune responses were induced. Moreover, mice receiving the vaccine were protected against tumor growth, demonstrating its efficacy in two tumor models, including the low immunogenic and aggressive B16/F10-Ova melanoma model. Remarkably, this approach was even effective in conditions of a late tumor challenge, i.e., 80days post-vaccination, between 88% (B16/F10-Ova melanoma) and 100% (EG7 thymoma) of mice remained tumor free. Thus, decorating AAV vector particles with antigens by capsid engineering represents a potent vaccine concept for applications in cancer immunotherapy. Its modular and versatile "plug-and-play" framework enables the use of tumor antigens of choice and the easy implementation of additional modifications to enhance immunogenicity further.

Fabry Disease: Current and Novel Therapeutic Strategies. A Narrative Review.

Fabry disease (FD) is an inherited lysosomal storage disorder, leading to multisystemic manifestations and causing significant morbidity and mortality. The aim of this narrative review is to present the current and novel therapeutic strategies in FD, including symptomatic and specific treatment options. A systematic literature search was conducted to identify relevant studies, including completed and ongoing randomized-controlled clinical trials (RCTs), prospective or retrospective cohort studies, case series and case reports that provided clinical data regarding FD treatment. A multidisciplinary symptomatic treatment is recommended for FD patients, personalized according to disease manifestations and their severity. During the last two decades, FD-specific treatments, including two enzyme-replacement-therapies (agalsidase alfa and agalsidase beta) and chaperone treatment with migalastat have been approved for use and allowed for symptoms' stabilization or even disease burden reduction. More therapeutic agents are currently under investigation. Substrate reduction therapies, including lucerastat and venglustat, have shown promising results in RCTs and may be used either as monotherapy or as complementary therapy to established enzymereplacement- therapies. More stable enzyme-replacement-therapy molecules that are associated with less adverse events and lower likelihood of neutralizing antibodies formation have also been developed. Ex-vivo and in-vivo gene therapy is being tested in animal models and pilot human clinical trials, with preliminary results showing a favorable safety and efficacy profile. The therapeutic landscape in FD appears to be actively expanding with more treatment options expected to become available in the near future, allowing for a more personalized approach in FD patients.

Aptamer-programmable adeno-associated viral vectors as a novel platform for cell-specific gene transfer

Adeno-associated viruses (AAVs) are commonly used for invivo gene therapy. Nevertheless, the wide tropism that characterizes these vectors limits specific targeting to a particular cell type or tissue. Here, we developed new chemically modified AAV vectors (Nε-AAVs) displaying a single site substitution on the capsid surface for post-production vector engineering through biorthogonal copper-free click chemistry. We were able to identify AAV vectors that would tolerate the unnatural amino acid substitution on the capsid without disrupting their packaging efficiency. We functionalized the Nε-AAVs through conjugation with DNA (AS1411) or RNA (E3) aptamers or with a folic acid moiety (FA). E3-, AS1411-, and FA-AAVs showed on average a 3- to 9-fold increase in transduction compared with their non-conjugated counterparts in different cancer cell lines. Using specific competitors, we established ligand-specific transduction. Invivo studies confirmed the selective uptake of FA-AAV and AS1411-AAV without off-target transduction in peripheral organs. Overall, the high versatility of these novel Nε-AAVs might pave the way to tailoring gene therapy vectors toward specific types of cells both for exvivo and invivo applications.

Effect of alcohol on productivity and quality of adeno-associated virus 2 in HEK293 cells

Investigation of enhancers to improve recombinant adeno-associated virus 2 (rAAV2) productivity by human embryonic kidney 293cells (HEK293) suspension culture showed that the addition of ethanol improved the productivity and packaged genome integrity of rAAV2. Further optimization showed that adding ethanol in the range of 0.09%-1.11% (v/v) during rAAV2 production effectively improved rAAV2 productivity and quality. In addition, ethanol addition improved cell viability. Furthermore, proteome and pathway analysis of the cells during rAAV2 production showed that the addition of ethanol resulted in the upregulation of pathways related to intercellular signaling, gene expression, cell morphology, intercellular maintenance, and others. In contrast, pathways related to cell death, immunity, and reactions to infection were downregulated. These changes in pathway regulation were responsible for the improvement in rAAV2 productivity, packaged genome integrity, and cell viability during rAAV2 production. The results of this study can be applied to the production of viral vectors for invivo gene therapy in an inexpensive and safe manner.

Development of alternative gene transfer techniques for exvivo and invivo gene therapy in a canine model.

IntroductionGene therapy have recently attracted much attention as a curative therapeutic option for inherited single gene disorders such as hemophilia. Hemophilia is a hereditary bleeding disorder caused by the deficiency of clotting activity of factor VIII (FVIII) or factor IX (FIX), and gene therapy for hemophilia using viral vector have been vigorously investigated worldwide. Toward further advancement of gene therapy for hemophilia, we have previously developed and validated the efficacy of novel two types of gene transfer technologies using a mouse model of hemophilia A. Here we investigated the efficacy and safety of the technologies in canine model. Especially, validations of technical procedures of the gene transfers for dogs were focused.MethodsGreen fluorescence protein (GFP) gene were transduced into normal beagle dogs by ex vivo and in vivo gene transfer techniques. For ex vivo gene transfer, blood outgrowth endothelial cells (BOECs) derived from peripheral blood of normal dogs were transduced with GFP gene using lentivirus vector, propagated, fabricated as cell sheets, then implanted onto the omentum of the same dogs. For in vivo gene transfer, normal dogs were subjected to GFP gene transduction with non-viral piggyBac vector by liver-targeted hydrodynamic injections.ResultsNo major adverse events were observed during the gene transfers in both gene transfer systems. As for ex vivo gene transfer, histological findings from the omental biopsy performed 4 weeks after implantation revealed the tube formation by implanted GFP-positive BOECs in the sub-adipose tissue layer without any inflammatory findings, and the detected GFP signals were maintained over 6 months. Regarding in vivo gene transfer, analyses of liver biopsy samples revealed more than 90% of liver cells were positive for GFP signals in the injected liver lobes 1 week after gene transfers, then the signals gradually declined overtime.ConclusionsTwo types of gene transfer techniques were successfully applied to a canine model, and the transduced gene expressions persisted for a long term. Toward clinical application for hemophilia patients, practical assessments of therapeutic efficacy of these techniques will need to be performed using a dog model of hemophilia and FVIII (or FIX) gene.

Rational Clinical Dose Selection of Adeno-Associated Virus-Mediated Gene Therapy Based on Allometric Principles.

One of the challenges in translational medicine is to select first-in-human doses of investigational drugs based on findings in preclinical studies. Despite substantial progress in the optimization of recombinant adeno-associated virus (AAV) vectors of in vivo gene therapy for treating various diseases, there remain significant limitations to the use of preclinical data to guide dose selection in clinical trials. Here we introduce a novel concept of gene efficiency factor (GEF) to describe the efficiency of the gene transfer system and describe and apply the concept of GEF in AAV-mediated in vivo gene transfer systems. We explore the utility of allometric scaling to translate GEF across species using AAV-mediated in vivo factor IX (FIX) gene therapy for hemophilia B and to demonstrate the use of GEF in predicting efficacious AAV vector doses in humans. We show for the first time that an allometric relationship exists for GEF of AAV-mediated in vivo gene therapy. Furthermore, we demonstrate the feasibility of using the allometric relationship of GEF to select efficacious first-in-human doses of virus-mediated invivo gene therapy. Based on our findings, allometry of GEF can be used to translate biological efficiency from animal studies to clinical studies and provide a rational basis of setting first-in-human doses for new virus-mediated invivo gene therapy products.

High-Efficiency, Selection-free Gene Repair in Airway Stem Cells from Cystic Fibrosis Patients Rescues CFTR Function in Differentiated Epithelia.

Cystic fibrosis (CF) is a monogenic disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Mortality in CF patients is mostly due to respiratory sequelae. Challenges with gene delivery have limited attempts to treat CF using invivo gene therapy, and low correction levels have hindered exvivo gene therapy efforts. We have used Cas9 and adeno-associated virus 6 to correct the ΔF508 mutation in readily accessible upper-airway basal stem cells (UABCs) obtained from CF patients. On average, we achieved 30%-50% allelic correction in UABCs and bronchial epithelial cells (HBECs) from 10 CF patients andobserved 20%-50% CFTR function relative to non-CF controls in differentiated epithelia. Furthermore, we successfully embedded the corrected UABCs on an FDA-approved porcine small intestinal submucosal membrane (pSIS), and they retained differentiation capacity. This study supports further development of genetically corrected autologous airway stem cell transplant as a treatment for CF.

In-Vivo Gene Therapy with Foamy Virus Vectors.

Foamy viruses (FVs) are nonpathogenic retroviruses that infect various animals including bovines, felines, nonhuman primates (NHPs), and can be transmitted to humans through zoonotic infection. Due to their non-pathogenic nature, broad tissue tropism and relatively safe integration profile, FVs have been engineered as novel vectors (foamy virus vector, FVV) for stable gene transfer into different cells and tissues. FVVs have emerged as an alternative platform to contemporary viral vectors (e.g., adeno associated and lentiviral vectors) for experimental and therapeutic gene therapy of a variety of monogenetic diseases. Some of the important features of FVVs include the ability to efficiently transduce hematopoietic stem and progenitor cells (HSPCs) from humans, NHPs, canines and rodents. We have successfully used FVV for proof of concept studies to demonstrate safety and efficacy following in-vivo delivery in large animal models. In this review, we will comprehensively discuss FVV based in-vivo gene therapy approaches established in the X-linked severe combined immunodeficiency (SCID-X1) canine model.

The Assembly-Activating Protein Promotes Stability and Interactions between AAV’s Viral Proteins to Nucleate Capsid Assembly

The adeno-associated virus (AAV) vector is a preferred delivery platform for invivo gene therapy. Natural and engineered variations of the AAV capsid affect a plurality of phenotypes relevant to gene therapy, including vector production and host tropism. Fundamental to these aspects is the mechanism of AAV capsid assembly. Here, the role of the viral co-factor assembly-activating protein (AAP) was evaluated in 12 naturally occurring AAVs and 9 putative ancestral capsid intermediates. The results demonstrate increased capsid protein stability and VP-VPinteractions in the presence of AAP. The capsid's dependence on AAP can be partly overcome bystrengthening interactions between monomers within the assembly, as illustrated by the transfer of a minimal motif defined by a phenotype-to-phylogeny mapping method. These findings suggest that the emergence of AAP within the Dependovirus genus relaxes structural constraints on AAV assembly in favor of increasing the degrees of freedom for the capsid to evolve.

Optimizing immune tolerizing regimens for in-vivo gene therapy

The mutation spectrum for the phenylalanine hydroxylase (PAH) gene was investigated in a cohort of 84 hyperphenylalaninemia (HPA) patients from Romania identified through newborn screening or neurometabolic investigations. Differential diagnosis identified 81 patients with classic PAH deficiency while 3 had tetrahydropterin-cofactor deficiency and/or remained uncertain due to insufficient specimen. PAH-genetic analysis included a combination of Sanger sequencing of exons and exon–intron boundaries, MLPA and NGS with genomic DNA, and cDNA analysis from immortalized lymphoblasts. A diagnostic efficiency of 99.4% was achieved, as for one allele (out of a total of 162 alleles) no mutation could be identified. The most prevalent mutation was p.Arg408Trp which was found in ~ 38% of all PKU alleles. Three novel mutations were identified, including the two missense mutations p.Gln226Lys and p.Tyr268Cys that were both disease causing by prediction algorithms, and the large genomic deletion EX6del7831 (c.509 + 4140_706 + 510del7831) that resulted in skipping of exon 6 based on PAH-cDNA analysis in immortalized lymphocytes. The genomic deletion was present in a heterozygous state in 12 patients, i.e. in ~ 8% of all the analyzed PKU alleles, and might have originated from a Romanian founder.

Abstract 5094: Precision therapeutic targeting of oncogenic FGFR3 fusions using CRISPR-Cas9 genome-editing in urothelial cancer

Abstract Introduction: Fibroblast-growth factor receptor 3 (FGFR3) fusions occur in up to 11 % of urothelial carcinomas. These fusions drive ligand-independent receptor dimerization and downstream oncogenic signaling and thus represent attractive targets for gene therapy. We hypothesized that CRISPR-Cas9 gene-editing can be used to disrupt these somatic oncogenic fusions in cancer cells. Methods: RT112 and SW780 urothelial cancer cell lines harboring genomic fusions of FGFR3 to TACC3, or BAIAP2L1, respectively were grown in cultures. The fusion sequences were confirmed by Sanger sequencing and the NGG protospacer adjacent motif necessary for guide RNA (gRNA) targeting were identified. Unique guide RNAs spanning the fusion sequences in each cell line were designed. An all-in-one lentiviral vector was designed to deliver both the gRNA and the Cas9 nuclease. A scrambled non-targeting gRNA vector was used as a control. Following transduction, genomic DNA was extracted and PCR was performed using primer sequences flanking the fusion sequences followed by targeted sequencing on the Illumina MiSeq platform. CrispRVariants and CRISPResso bioinformatic tools were used to determine editing efficiency. Results: In SW780 cells transduced with the FGFR3-BAIAP2L1 fusion-targeting gRNA vector, 64229/207425 (23.6%) reads harbored CRISPR-induced edits of the fusion sequence compared to only 2009/313553(0.6%) reads in the scrambled gRNA vector-transduced cells. Similarly, in RT112 cells transduced with the FGFR3-TACC3 fusion-targeting gRNA vector, 74292/448381 reads (14.2%) contained random indels consistent with CRISPR-induced edits compared to 17785/428035 reads (4.0%) in RT112s cells transduced with the scrambled gRNA vector. In the SW780 cell line, the most common indel in CRISPR-edited fusions sequences was a deletion of a cytosine occurring in 40% of edited reads. The second most common indel was an extra cytosine insertion and the third most common was a 13-base deletion spanning the fusion point. In RT112 cell line, the most common indel was an insertion of a cytosine in 13% of reads. Sanger sequencing of the fusion sequences from SW780 single cell clones confirmed Miseq results. Experiments determining the effects of these indels on the function of the fusion protein and on the cancer phenotype are ongoing. Conclusions: To our knowledge, this is the first report of CRISPR/Cas9 use as a therapeutic strategy for targeting oncogenic fusions. Because the fusion sequences are unique to cancer cells, this approach can be potentially used for in-vivo gene therapy for urothelial carcinomas harboring FGFR3 fusions with no off-target edits in normal cells. This approach represents a significant advance towards precision gene-therapy by specifically targeting fusions sequences unique to each patient’s cancer. We envision extending this approach to several solid and hematological malignancies driven by oncogenic fusions. Citation Format: Bishoy M. Faltas, Rebecca Meyer, Ethan Shelkey, Charles J. Murphy, Olivier Elemento, Mark A. Rubin. Precision therapeutic targeting of oncogenic FGFR3 fusions using CRISPR-Cas9 genome-editing in urothelial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5094. doi:10.1158/1538-7445.AM2017-5094

Progress in gene therapy for primary immunodeficiencies using lentiviral vectors.

This review gives an overview over the most recent progress in the field of lentiviral gene therapy for primary immunodeficiencies (PIDs). The history and state-of-the-art of lentiviral vector development are summarized and the recent advancements for a number of selected diseases are reviewed in detail. Past retroviral vector trials for these diseases, the most recent improvements of lentiviral vector platforms and their application in preclinical development as well as ongoing clinical trials are discussed. Main focus is on the preclinical studies and clinical trials for the treatment of Wiskott-Aldrich syndrome, chronic granulomatous disease, adenosine deaminase deficient severe combined immunodeficiency (ADA-SCID) and X-linked severe combined immunodeficiency with lentiviral gene therapy. Gene therapy for PIDs is an effective treatment, providing potential long-term clinical benefit for affected patients. Substantial progress has been made to make lentiviral gene therapy platforms available for a number of rare genetic diseases. Although many ongoing gene therapy trials are based on ex-vivo approaches with autologous hematopoietic stem cells, other approaches such as in-vivo gene therapy or gene-repair platforms might provide further advancement for certain PIDs.

Recent advances in ocular gene therapy

Ocular gene therapy has made significant advances due to improvements in viral vector delivery methods. Recent clinical trials for the treatment of Leber's congenital amaurosis have provided evidence for successful in-vivo gene therapy in humans. Gene therapy for ocular disease has been under investigation just over 15 years. Recently, the first human gene therapy trials for retinal degeneration were undertaken with encouraging preliminary safety and efficacy findings. Building on previous success in reversing blindness in animal models of Leber's congenital amaurosis, several groups proceeded with adeno-associated virus-mediated gene replacement. Many of the humans demonstrated increases in light sensitivity and in visual acuity. Subjective improvements in vision were corroborated in some cases by objective tests such as pupillary light response and nystagmography. Although much of the work in ocular gene therapy has involved retinal applications, significant progress has been seen in other aspects of ophthalmology. Ongoing human clinical trials support the safety and efficacy of adeno-associated virus-mediated gene therapy for retinal disease. These and other studies will establish the foundation for methodology to treat additional ocular diseases using gene therapy strategies.

Gene therapy for Parkinson's disease: early data

Parkinson's disease is the second most common neurodegenerative disorder, affecting at least 1% of people above the age of 65 years, a proportion that increases with age. The introduction of replacement therapy with levodopa and, more recently, various adjunct drugs has substantially improved survival and quality of life. However, these therapies are associated with several long-term complications that can limit the tolerated dose. None of the available drugs has a convincing effect on the inexorable progression of the disease. Surgical approaches, such as deep-brain stimulation of the subthalamic nucleus, are effective 1 Deuschl G Schade-Brittinger C Krack P et al. A randomized trial of deep-brain stimulation for Parkinson's disease. N Engl J Med. 2006; 355: 896-908 Crossref PubMed Scopus (2018) Google Scholar but technically complex, and continuing care from an expert team is needed. Although there is interest in cell-based therapies, double-blind controlled trials of human fetal mesencephalic transplantation were disappointing. 2 Freed CR Greene PE Breeze RE et al. Transplantation of embryonic dopamine neurons for severe Parkinson's disease. N Engl J Med. 2001; 344: 710-719 Crossref PubMed Scopus (1968) Google Scholar , 3 Olanow CW Goetz CG Kordower JH et al. A double-blind controlled trial of bilateral fetal nigral transplantation in Parkinson's disease. Ann Neurol. 2003; 54: 403-414 Crossref PubMed Scopus (1244) Google Scholar Several gene-therapy approaches have been suggested or are in development, ranging from those that attempt to restore dopamine synthetic capacity 4 Bankiewicz KS Forsayeth J Eberling JL et al. Long-term clinical improvement in MPTP-lesioned primates after gene therapy with AAV-hAADC. Mol Ther. 2006; 14: 564-570 Summary Full Text Full Text PDF PubMed Scopus (235) Google Scholar , 5 Pardridge WM Tyrosine hydroxylase replacement in experimental Parkinson's disease with transvascular gene therapy. NeuroRx. 2005; 2: 129-138 Crossref PubMed Scopus (83) Google Scholar to those that protect against cell death with trophic factors, 6 Kordower JH Herzog CD Dass B et al. Delivery of neurturin by AAV2 (CERE-120)-mediated gene transfer provides structural and functional neuroprotection and neurorestoration in MPTP-treated monkeys. Ann Neurol. 2006; 60: 706-715 Crossref PubMed Scopus (216) Google Scholar to molecules that interfere with the aberrant protein aggregation in Parkinson's disease. 7 Lo Bianco C Schneider BL Bauer M et al. Lentiviral vector delivery of parkin prevents dopaminergic degeneration in an α-synuclein rat model of Parkinson's disease. Proc Natl Acad Sci USA. 2004; 101: 17510-17515 Crossref PubMed Scopus (270) Google Scholar , 8 Mochizuki H Yamada M Mizuno Y Parkin gene therapy for α-synucleinopathy: a rat model of Parkinson's disease. Parkinsonism Relat Disord. 2006; 12: S107-S109 Summary Full Text Full Text PDF Scopus (6) Google Scholar Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson's disease: an open label, phase I trialAAV-GAD gene therapy of the subthalamic nucleus is safe and well tolerated by patients with advanced Parkinson's disease, suggesting that in-vivo gene therapy in the adult brain might be safe for various neurodegenerative diseases. Full-Text PDF

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

Dopaminergic neuronal loss in Parkinson's disease leads to changes in the circuitry of the basal ganglia, such as decreased inhibitory GABAergic input to the subthalamic nucleus. We aimed to measure the safety, tolerability, and potential efficacy of transfer of glutamic acid decarboxylase (GAD) gene with adeno-associated virus (AAV) into the subthalamic nucleus of patients with Parkinson's disease. We did an open label, safety and tolerability trial of unilateral subthalamic viral vector (AAV-GAD) injection in 11 men and 1 woman with Parkinson's disease (mean age 58.2, SD=5.7 years). Four patients received low-dose, four medium-dose, and four high-dose AAV-GAD at New York Presbyterian Hospital. Inclusion criteria consisted of Hoehn and Yahr stage 3 or greater, motor fluctuations with substantial off time, and age 70 years or less. Patients were assessed clinically both off and on medication at baseline and after 1, 3, 6, and 12 months at North Shore Hospital. Efficacy measures included the Unified Parkinson's Disease Rating Scale (UPDRS), scales of activities of daily living (ADL), neuropsychological testing, and PET imaging with 18F-fluorodeoxyglucose. The trial is registered with the ClinicalTrials.gov registry, number NCT00195143. All patients who enrolled had surgery, and there were no dropouts or patients lost to follow-up. There were no adverse events related to gene therapy. Significant improvements in motor UPDRS scores (p=0.0015), predominantly on the side of the body that was contralateral to surgery, were seen 3 months after gene therapy and persisted up to 12 months. PET scans revealed a substantial reduction in thalamic metabolism that was restricted to the treated hemisphere, and a correlation between clinical motor scores and brain metabolism in the supplementary motor area. AAV-GAD gene therapy of the subthalamic nucleus is safe and well tolerated by patients with advanced Parkinson's disease, suggesting that in-vivo gene therapy in the adult brain might be safe for various neurodegenerative diseases.