Therapeutic Transgene Research Articles

An Inducible Promoter Responsive to Different Porphyrinogenic Stimuli Improves Gene Therapy Vectors for Acute Intermittent Porphyria.

Porphobilinogen deaminase (PBGD) gene therapy represents a promising therapeutic option for acute intermittent porphyria (AIP) patients suffering recurrent acute attacks. A first-in-human Phase I clinical trial confirmed the safety and tolerability of adeno-associated virus (AAV)-AAT-PBGD gene therapy, but higher doses and/or more efficient vectors are needed to achieve therapeutic expression of the transgene. This study assayed the insertion into the promoter of a short enhancer element able to induce transgene expression during exposure to endogenous and exogenous stimuli related to the pathology of the disease. The inclusion in tandem of two elements of the minimal functional sequence of human δ-aminolevulinic acid synthase drug-responsive enhancing sequence (ADRES) positioned upstream of the promoter strongly induced transgene expression in the presence of estrogens, starvation, and certain drugs known to trigger attacks in porphyria patients. The inclusion of two ADRES motives in an AAV vector improved therapeutic efficacy, reducing 10-fold the effective dose in AIP mice. In conclusion, the inclusion of specific enhancer elements in the promoter of gene therapy vectors for AIP was able to overexpress the therapeutic transgene when it is most needed, at the time when porphyrinogenic factors increase the demand for hepatic heme and precipitate acute porphyria attacks.

Comparative Analysis of Gene Therapy Systems Expressing Two Oncotherapeutic Genes under Control of a Single Promotor

To improve the safety profile and specificity in cancer gene therapy applications, а set of singleplasmid vectors for expression of suicide thymidine kinase gene of the herpes simplex virus (HSVtk) and mouse OX40L gene (m0X40L) involved in regulation of immune response was constructed. Therapeutic transgene expression was directed by a strong constitutive promoter of human cytomegalovirus (CMV), while translation occurred on an RNA template due to an encephalomyocarditis virus internal ribosome entry site (construct CMV-HSVtk-IRES-mOX40L) or 2A-peptide of Porcine Teschovirus-1 (construct CMV-HSVtk-2AmOX40L) inserted between them. The constructs obtained were functionally validated by transient transfection of C26 cells (mouse colon carcinoma). Both bicystronic vectors demonstrated strong cytotoxic activity and produced cytotoxic protein HSVtk at the similar level. To analyze efficiency of mOX40L expression, C26 transfected cells were immunofluorescence-labelled with anti-OX40L-antibodies conjugated with phycoerythrin following flow cytometric analysis. Production of mOX40L was higher in the case of CMV-HSVtk- 2A-mOX40L. Better performance of this expression vector was correlated with our data. Previously, we have demonstrated that a higher level of transgene expression was exhibited by cells transfected with the vector containing the 2A peptide sequence. Thus, transfection with the vector based on the 2A peptide sequence is optimal for achieving high efficiency of expression of both oncotherapeutic genes.

Efficient Therapeutic Protein Expression Using Retroviral Replicating Vector with 2A Peptide in Cancer Models.

Toca 511, a retroviral replicating vector (RRV), uses an internal ribosomal entry site (IRES) to express an optimized yeast cytosine deaminase (yCD2), which converts 5-fluorocytosine to 5-fluorouracil. This configuration is genetically stable in both preclinical mouse models and human clinical trials. However, the use of IRES (∼600 bp) restricts choices of therapeutic transgenes due to limits in RRV genome size. This study replaced IRES with 2A peptides derived from picornaviruses with or without a GSG linker. The data show that GSG-linked 2A (g2A) peptide resulted in higher polyprotein separation efficiency than non-GSG linked 2A peptide. The study also shows that RRV can tolerate insertion of two separate 2A peptides to allow expression of two transgenes without compromising the assembly and function of the virus in addition to insertion of a single 2A peptide to confirm genetic stability with yCD2, green fluorescent protein, and HSV-1 thymidine kinase. In a parallel comparison of the RRV-IRES-yCD2 and RRV-g2A-yCD2 configurations, the study shows the yCD2 protein expressed from RRV-g2A-yCD2 has higher activity, resulting in a higher survival benefit in an intracranial tumor mouse model. These data enable a wider range of potential product candidates that could be developed using the RRV platform.

Preclinical and clinical advances in transposon-based gene therapy.

Transposons derived from Sleeping Beauty (SB), piggyBac (PB), or Tol2 typically require cotransfection of transposon DNA with a transposase either as an expression plasmid or mRNA. Consequently, this results in genomic integration of the potentially therapeutic gene into chromosomes of the desired target cells, and thus conferring stable expression. Non-viral transfection methods are typically preferred to deliver the transposon components into the target cells. However, these methods do not match the efficacy typically attained with viral vectors and are sometimes associated with cellular toxicity evoked by the DNA itself. In recent years, the overall transposition efficacy has gradually increased by codon optimization of the transposase, generation of hyperactive transposases, and/or introduction of specific mutations in the transposon terminal repeats. Their versatility enabled the stable genetic engineering in many different primary cell types, including stem/progenitor cells and differentiated cell types. This prompted numerous preclinical proof-of-concept studies in disease models that demonstrated the potential of DNA transposons for ex vivo and in vivo gene therapy. One of the merits of transposon systems relates to their ability to deliver relatively large therapeutic transgenes that cannot readily be accommodated in viral vectors such as full-length dystrophin cDNA. These emerging insights paved the way toward the first transposon-based phase I/II clinical trials to treat hematologic cancer and other diseases. Though encouraging results were obtained, controlled pivotal clinical trials are needed to corroborate the efficacy and safety of transposon-based therapies.

Mapping the Structural Determinants Required for AAVrh.10 Transport across the Blood-Brain Barrier.

Effective gene delivery to the CNS by intravenously administered adeno-associated virus (AAV) vectors requires crossing the blood-brain barrier (BBB). To achieve therapeutic CNS transgene expression, high systemic vector doses are often required, which poses challenges such as scale-up costs and dose-dependent hepatotoxicity. To improve the specificity and efficiency of CNS gene transfer, a better understanding of the structural features that enable AAV transit across the BBB is needed. We generated a combinatorial domain swap library using AAV1, a serotype that does not traverse the vasculature, and AAVrh.10, which crosses the BBB in mice. We then screened individual variants by phylogenetic and structural analyses and subsequently conducted systemic characterization in mice. Using this approach, we identified key clusters of residues on the AAVrh.10 capsid that enabled transport across the brain vasculature and widespread neuronal transduction in mice. Through rational design, we mapped a minimal footprint from AAVrh.10, which, when grafted onto AAV1, confers the aforementioned CNS phenotype while diminishing vascular and hepatic transduction through an unknown mechanism. Functional mapping of this capsid surface footprint provides a roadmap for engineering synthetic AAV capsids for efficient CNS gene transfer with an improved safety profile.

Enhanced liver gene transfer and evasion of preexisting humoral immunity with exosome-enveloped AAV vectors

AbstractResults from clinical trials of liver gene transfer for hemophilia demonstrate the potential of the adeno-associated virus (AAV) vector platform. However, to achieve therapeutic transgene expression, in some cases high vector doses are required, which are associated with a higher risk of triggering anti-capsid cytotoxic T-cell responses. Additionally, anti-AAV preexisting immunity can prevent liver transduction even at low neutralizing antibody (NAb) titers. Here, we describe the use of exosome-associated AAV (exo-AAV) vectors as a robust liver gene delivery system that allows the therapeutic vector dose to be decreased while protecting from preexisting humoral immunity to the capsid. The in vivo efficiency of liver targeting of standard AAV8 or AAV5 and exo-AAV8 or exo-AAV5 vectors expressing human coagulation factor IX (hF.IX) was evaluated. A significant enhancement of transduction efficiency was observed, and in hemophilia B mice treated with 4 × 1010 vector genomes per kilogram of exo-AAV8 vectors, a staggering ∼1 log increase in hF.IX transgene expression was observed, leading to superior correction of clotting time. Enhanced liver expression was also associated with an increase in the frequency of regulatory T cells in lymph nodes. The efficiency of exo- and standard AAV8 vectors in evading preexisting NAbs to the capsid was then evaluated in a passive immunization mouse model and in human sera. Exo-AAV8 gene delivery allowed for efficient transduction even in the presence of moderate NAb titers, thus potentially extending the proportion of subjects eligible for liver gene transfer. Exo-AAV vectors therefore represent a platform to improve the safety and efficacy of liver-directed gene transfer.

Cowpox Virus: A New and Armed Oncolytic Poxvirus

Oncolytic virus therapy has recently been recognized as a promising new therapeutic approach for cancer treatment. In this study, we are proposing for the first time to evaluate the in vitro and in vivo oncolytic capacities of the Cowpox virus (CPXV). To improve the tumor selectivity and oncolytic activity, we developed a thymidine kinase (TK)-deleted CPXV expressing the suicide gene FCU1, which converts the non-toxic prodrug 5-fluorocytosine (5-FC) into cytotoxic 5-fluorouracil (5-FU) and 5-fluorouridine-5′-monophosphate (5-FUMP). This TK-deleted virus replicated efficiently in human tumor cell lines; however, it was notably attenuated in normal primary cells, thus displaying a good therapeutic index. Furthermore, this new recombinant poxvirus rendered cells sensitive to 5-FC. In vivo, after systemic injection in mice, the TK-deleted variant caused significantly less mortality than the wild-type strain. A biodistribution study demonstrated high tumor selectivity and low accumulation in normal tissues. In human xenograft models of solid tumors, the recombinant CPXV also displayed high replication, inducing relevant tumor growth inhibition. This anti-tumor effect was improved by 5-FC co-administration. These results demonstrated that CPXV is a promising oncolytic vector capable of expressing functional therapeutic transgenes.

Transposons: Moving Forward from Preclinical Studies to Clinical Trials.

Transposons have emerged as promising vectors for gene therapy that can potentially overcome some of the limitations of commonly used viral vectors. Transposons stably integrate into the target cell genome, enabling persistent expression of therapeutic genes. Transposons have evolved from being used as basic tools in biomedical research to bona fide therapeutics. Currently, the most promising transposons for gene therapy applications are derived from Sleeping Beauty (SB) or piggyBac (PB). Stable transposition requires co-delivery of the transposon DNA with the corresponding transposase gene, mRNA, or protein. Stable transposition efficiency can be substantially increased by using "next-generation" transposon systems that combine codon-usage optimization with hyper-activating mutations in the SB or PB transposases. By virtue of their relatively large capacity, gene therapy applications with relatively large therapeutic transgenes, such as full-length dystrophin, can now be envisaged. The authors and others have shown that efficient and stable gene transfer can be achieved with these next-generation transposons in several clinically relevant primary cells, such as CD34+ hematopoietic stem/progenitor cells, T cells, and mesenchymal and myogenic stem/progenitor cells that are amenable for ex vivo transfection. Alternatively, in vivo transposon gene delivery has been explored using non-viral vectors or nanoparticles or in combination with viral vectors. The therapeutic potential of these SB- and PB-based transposons has been demonstrated in preclinical models that mimic the cognate human diseases. However, there are still challenges impeding clinical translation of transposons pertaining mainly to the typical limiting efficiencies of most non-viral transfection methods and the intrinsic DNA toxicity. Nevertheless, it is particularly encouraging that transposons have now been used in gene therapy clinical trials. In particular, transposon-engineered T cells expressing chimeric antigen receptors are starting to yield promising results in patients with hematological malignancies.

The CpG-sites of the CBX3 ubiquitous chromatin opening element are critical structural determinants for the anti-silencing function

Suppression of therapeutic transgene expression from retroviral gene therapy vectors by epigenetic defence mechanisms represents a problem that is particularly encountered in pluripotent stem cells (PSCs) and their differentiated progeny. Transgene expression in these cells, however, can be stabilised by CpG-rich ubiquitous chromatin opening elements (UCOEs). In this context we recently demonstrated profound anti-silencing properties for the small (679 bp) CBX3-UCO element and we now confirmed this observation in the context of the defined murine chromosomal loci ROSA26 and TIGRE. Moreover, since the structural basis for the anti-silencing activity of UCOEs has remained poorly defined, we interrogated various CBX3 subfragments in the context of lentiviral vectors and murine PSCs. We demonstrated marked though distinct anti-silencing activity in the pluripotent state and during PSC-differentiation for several of the CBX3 subfragments. This activity was significantly correlated with CpG content as well as endogenous transcriptional activity. Interestingly, also a scrambled CBX3 version with preserved CpG-sites retained the anti-silencing activity despite the lack of endogenous promoter activity. Our data therefore highlight the importance of CpG-sites and transcriptional activity for UCOE functionality and suggest contributions from different mechanisms to the overall anti-silencing function of the CBX3 element.

Dendronized Semiconducting Polymer as Photothermal Nanocarrier for Remote Activation of Gene Expression

AbstractRegulation of transgene systems is needed to develop innovative medicines. However, noninvasive remote control of gene expression has been rarely developed and remains challenging. We herein synthesize a near‐infrared (NIR) absorbing dendronized semiconducting polymer (DSP) and utilize it as a photothermal nanocarrier not only to efficiently deliver genes but also to spatiotemporally control gene expression in conjunction with heat‐inducible promoter. DSP has a high photothermal conversion efficiency (44.2 %) at 808 nm, permitting fast transduction of NIR light into thermal signals for intracellular activation of transcription. Such a DSP‐mediated remote activation can rapidly and safely result in 25‐ and 4.5‐fold increases in the expression levels of proteins in living cells and mice, respectively. This study thus provides a promising approach to optically regulate transgene systems for on‐demand therapeutic transgene dosing.

Dendronized Semiconducting Polymer as Photothermal Nanocarrier for Remote Activation of Gene Expression.

Regulation of transgene systems is needed to develop innovative medicines. However, noninvasive remote control of gene expression has been rarely developed and remains challenging. We herein synthesize a near-infrared (NIR) absorbing dendronized semiconducting polymer (DSP) and utilize it as a photothermal nanocarrier not only to efficiently deliver genes but also to spatiotemporally control gene expression in conjunction with heat-inducible promoter. DSP has a high photothermal conversion efficiency (44.2 %) at 808 nm, permitting fast transduction of NIR light into thermal signals for intracellular activation of transcription. Such a DSP-mediated remote activation can rapidly and safely result in 25- and 4.5-fold increases in the expression levels of proteins in living cells and mice, respectively. This study thus provides a promising approach to optically regulate transgene systems for on-demand therapeutic transgene dosing.

Abstract 4556: MEDI5395: An armed oncolytic Newcastle disease virus

Abstract Oncolytic viruses are live, replication-competent viruses that infect and/or replicate selectively in tumour cells leading to the destruction of the infected cell. Cell lysis occurs as a natural consequence of the viral life cycle and released virions can infect and kill neighbouring tumour cells leading to an amplified therapeutic effect. Oncolysis has the added benefit of releasing multiple tumour antigens that may further induce an immune-mediated therapeutic response. Newcastle Disease Virus (NDV) is an avian paramyxovirus, which has proven safety and demonstrated efficacy against a variety of preclinical cancer models and in PhI clinical studies as an oncolytic agent, oncolysate or whole cell vaccine. Using reverse genetics, we have generated a recombinant strain of NDV that overcomes environmental and regulatory concerns uncoupling oncolytic potency and avian pathogenicity. Furthermore we have enhanced the immune modulatory properties of NDV by engineering the virus to express granulocyte/macrophage colony-stimulating factor (GM-CSF). We have evaluated the biological characteristics of recNDVGM-CSF (MEDI5395) in vivo and in vitro. MEDI5395 selectively replicates in and kills a wide variety of human and mouse tumour cell lines. Additionally infection of cancer cells with MEDI5395 results in the increased production and secretion of pro-inflammatory cytokines and chemokines which are able to recruit mediators of both the innate and adaptive immune responses. MEDI5395 is a potent activator of the type I interferon response. In vivo, using a range of syngeneic and xenograft models we have demonstrated that NDV treatment has robust anti-tumour activity. In a HT1080 fibrosarcoma xenograft model a single administration (intra-tumoural or systemic) was able to cure 80% of tumour bearing mice. In syngeneic mouse tumour models, which support minimal viral replication MEDI5395 treatment causes significant changes in the local immune suppressive microenvironment and results in long-lasting anti-tumour immune responses. These responses are further enhanced in models that permit greater replication and also when combined with immune checkpoint blockade. The inherent properties of NDV (self-propagation, tumour-selective replication, and immunostimulatory properties) coupled with the ability to genetically engineer NDV to express therapeutic transgenes may provide a multi-modal attack on the tumour, delivering greater benefit to patients. Citation Format: Danielle Carroll, James Harper, Travers Jon, Shannon Burke, Ruth Franks, Christel Navarro, Xing Cheng, Robert Wilkinson, Hong Jin. MEDI5395: An armed oncolytic Newcastle disease virus [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 4556. doi:10.1158/1538-7445.AM2017-4556

Current Immunotherapeutic Strategies to Enhance Oncolytic Virotherapy.

Oncolytic viruses (OV) represent a promising strategy to augment the spectrum of cancer therapeutics. For efficacy, they rely on two general mechanisms: tumor-specific infection/cell-killing, followed by subsequent activation of the host’s adaptive immune response. Numerous OV genera have been utilized in clinical trials, ultimately culminating in the 2015 Food and Drug Administration approval of a genetically engineered herpes virus, Talminogene laherparepvec (T-VEC). It is generally accepted that OV as monotherapy have only modest clinical efficacy. However, due to their ability to elicit specific antitumor immune responses, they are prime candidates to be paired with other immune-modulating strategies in order to optimize therapeutic efficacy. Synergistic strategies to enhance the efficacy of OV include augmenting the host antitumor response through the insertion of therapeutic transgenes such as GM-CSF, utilization of the prime-boost strategy, and combining OV with immune-modulatory drugs such as cyclophosphamide, sunitinib, and immune checkpoint inhibitors. This review provides an overview of these immune-based strategies to improve the clinical efficacy of oncolytic virotherapy.

Development of a versatile oncolytic virus platform for local intra-tumoural expression of therapeutic transgenes.

Oncolytic viruses which infect and kill tumour cells can also be genetically modified to express therapeutic genes that augment their anti-cancer activities. Modifying oncolytic viruses to produce effective cancer therapies is challenging as encoding transgenes often attenuates virus activity or prevents systemic delivery in patients due to the risk of off-target expression of transgenes in healthy tissues. To overcome these issues we aimed to generate a readily modifiable virus platform using the oncolytic adenovirus, enadenotucirev. Enadenotucirev replicates in human tumour cells but not cells from healthy tissues and can be delivered intravenously because it is stable in human blood. Here, the enadenotucirev genome was used to generate plasmids into which synthesised transgene cassettes could be directly cloned in a single step reaction. The platform enabled generation of panels of reporter viruses to identify cloning sites and transgene cassette designs where transgene expression could be linked to the virus life cycle. It was demonstrated using these viruses that encoded transgene proteins could be successfully expressed in tumour cells in vitro and tumours in vivo. The expression of transgenes did not impact either the oncolytic activity or selective properties of the virus. The effectiveness of this approach as a drug delivery platform for complex therapeutics was demonstrated by inserting multiple genes in the virus genome to encode full length anti-VEGF antibodies. Functional antibody could be synthesised and secreted from infected tumour cells without impacting the activity of the virus particle in terms of oncolytic potency, manufacturing yields or selectivity for tumour cells. In vivo, viral particles could be efficaciously delivered intravenously to disseminated orthotopic tumours.

Engineered HSV vector achieves safe long-term transgene expression in the central nervous system

Previously we reported a new series of highly defective herpes simplex virus type 1 (HSV-1) vectors that were functionally devoid of all viral immediately early (IE) genes, resulting in virtual absence of viral gene expression. Nevertheless, a reporter gene cassette inserted into the vector flanked by boundary elements from the viral latency locus showed high, persistent reporter gene activity in non-neuronal cells while an independent expression cassette inserted into a deleted ICP4 locus remained almost silent. In contrast to non-neuronal cells, we show here that the ICP4 locus cassette permitted robust reporter gene expression in a diversity of neurons following stereotactic injection of different rat brain regions; transgene expression in the hippocampus lasted up to 6 months and was essentially restricted to neurons. No evidence of neuronal cell toxicity or induction of inflammatory cell infiltrates was observed. An independent reporter gene cassette located in an intergenic region remained silent, indicating that the transgene promoter and/or insertion site are critical for sustained expression. These findings suggest the suitability of this vector for therapeutic intervention into diseases of the central nervous system that require the expression of large and/or multiple therapeutic transgenes.

Enhanced Vector Design for Cancer Gene Therapy with Hierarchical Enhancement of Therapeutic Transgene Expression.

A set of vectors for Cre recombinase-dependent expression of the hybrid suicidal FCU1 transgene was constructed, including a two-plasmid system wherein the FCU1 and Cre transgenes reside in separate vectors, and single-plasmid variants in which a single plasmid bears both transgenes. To improve the safety profile and specificity in cancer gene therapy applications, as well as to ensure stable propagation of plasmids in bacterial cells, the Cre/LoxP system components were optimized. A bicistronic vector with the Cre expression cassette placed between the LoxP sites unidirectionally with FCU1 cDNA resulted in higher therapeutic efficiency compared with the double-plasmid system in an enzyme-prodrug suicide cancer gene therapy scheme. Therefore, the feasibility of a single-plasmid approach in the development of cancer gene therapy with hierarchical enhancement of therapeutic transgene expression has been demonstrated.

Long-Term Efficacy and Safety of Insulin and Glucokinase Gene Therapy for Diabetes: 8-Year Follow-Up in Dogs

Diabetes is a complex metabolic disease that exposes patients to the deleterious effects of hyperglycemia on various organs. Achievement of normoglycemia with exogenous insulin treatment requires the use of high doses of hormone, which increases the risk of life-threatening hypoglycemic episodes. We developed a gene therapy approach to control diabetic hyperglycemia based on co-expression of the insulin and glucokinase genes in skeletal muscle. Previous studies proved the feasibility of gene delivery to large diabetic animals with adeno-associated viral (AAV) vectors. Here, we report the long-term (∼8 years) follow-up after a single administration of therapeutic vectors to diabetic dogs. Successful, multi-year control of glycemia was achieved without the need of supplementation with exogenous insulin. Metabolic correction was demonstrated through normalization of serum levels of fructosamine, triglycerides, and cholesterol and remarkable improvement in the response to an oral glucose challenge. The persistence of vector genomes and therapeutic transgene expression years after vector delivery was documented in multiple samples from treated muscles, which showed normal morphology. Thus, this study demonstrates the long-term efficacy and safety of insulin and glucokinase gene transfer in large animals and especially the ability of the system to respond to the changes in metabolic needs as animals grow older.

Refining strategies to translate genome editing to the clinic.

Recent progress in developing programmable nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas nucleases, have paved the way for gene editing to enter clinical practice. This translation is a result of combining high nuclease activity with high specificity and successfully applying this technology in various preclinical disease models, including infectious disease, primary immunodeficiencies, hemoglobinopathies, hemophilia and muscular dystrophy. Several clinical gene-editing trials, both ex vivo and in vivo, have been initiated in the past 2 years, including studies that aim to knockout genes as well as to add therapeutic transgenes. Here we discuss the advances made in the gene-editing field in recent years, and specify priorities that need to be addressed to expand therapeutic genome editing to further disease entities.

Enhancing titres of therapeutic viral vectors using the transgene repression in vector production (TRiP) system

A key challenge in the field of therapeutic viral vector/vaccine manufacturing is maximizing production. For most vector platforms, the ‘benchmark’ vector titres are achieved with inert reporter genes. However, expression of therapeutic transgenes can often adversely affect vector titres due to biological effects on cell metabolism and/or on the vector virion itself. Here, we exemplify the novel ‘Transgene Repression In vector Production’ (TRiP) system for the production of both RNA- and DNA-based viral vectors. The TRiP system utilizes a translational block of one or more transgenes by employing the bacterial tryptophan RNA-binding attenuation protein (TRAP), which binds its target RNA sequence close to the transgene initiation codon. We report enhancement of titres of lentiviral vectors expressing Cyclo-oxygenase-2 by 600-fold, and adenoviral vectors expressing the pro-apoptotic gene Bax by >150,000-fold. The TRiP system is transgene-independent and will be a particularly useful platform in the clinical development of viral vectors expressing problematic transgenes.

Ex vivo gene therapy for the treatment of neurological disorders.

Ex vivo gene therapy involves the genetic modification of cells outside of the body to produce therapeutic factors and their subsequent transplantation back into patients. Various cell types can be genetically engineered. However, with the explosion in stem cell technologies, neural stem/progenitor cells and mesenchymal stem cells are most often used. The synergy between the effect of the new cell and the additional engineered properties can often provide significant benefits to neurodegenerative changes in the brain. In this review, we cover both preclinical animal studies and clinical human trials that have used ex vivo gene therapy to treat neurological disorders with a focus on Parkinson's disease, Huntington's disease, Alzheimer's disease, ALS, and stroke. We highlight some of the major advances in this field including new autologous sources of pluripotent stem cells, safer ways to introduce therapeutic transgenes, and various methods of gene regulation. We also address some of the remaining hurdles including tunable gene regulation, in vivo cell tracking, and rigorous experimental design. Overall, given the current outcomes from researchers and clinical trials, along with exciting new developments in ex vivo gene and cell therapy, we anticipate that successful treatments for neurological diseases will arise in the near future.