Gene Therapy Studies Research Articles

STEM-01. CHARACTERIZATION OF ORTHOTOPIC GLIOBLASTOMA MODEL FOR THE GENE-AND CELL THERAPY

Abstract Intrinsic heterogeneous and infiltrative nature of glioblastoma cells to the adjacent normal brain parenchyma are the main obstacles to the treatment of glioblastoma multiforme (GBM). The infiltrative nature of GBM increases following various treatments, which often leads to tumor recurrence. U87 cell line, a commonly used as a GBM cell line till recently, has appeared inappropriate for GBM model. Thus, we established an orthotopic GBM model using another cell line, LN229. Here, we discuss the infiltrative nature of LN229 in the mouse brain and their interaction with adjacent brain microenvironment. We also show that the LN229-driven brain tumor model is appropriate for the study of mesenchymal stem cell-mediated gene therapy using deliver a bacterial suicide gene, cytosine deaminase (CD).

Low-Dose Busulfan Reduces Human CD34+ Cell Doses Required for Engraftment in c-kit Mutant Immunodeficient Mice

Humanized animal models are central to efforts aimed at improving hematopoietic stem cell (HSC) transplantation with or without genetic modification. Human cell engraftment is feasible in immunodeficient mice; however, high HSC doses and conditioning limit broad use of xenograft models. We assessed human CD45+ chimerism after transplanting varying doses of human CD34+ HSCs (2 × 105 to 2 × 106 cells/mouse) with or without busulfan (BU) pretransplant conditioning in c-kit mutant mice that do not require conditioning (non-obese diabetic [NOD]/B6/severe combined immunodeficiency [SCID]/ interleukin-2 receptor gamma chain null (IL-2rγ−/−) KitW41/W41 [NBSGW]). We then tested a range of BU (5–37.5 mg/kg) using 2 × 105 human CD34+ cells. Glycophorin-A erythrocyte chimerism was assessed after murine macrophage depletion using clodronate liposomes. We demonstrated successful long-term engraftment of human CD34+ cells at all cell doses in this model, and equivalent engraftment using 10-fold less CD34+ cells with the addition of BU conditioning. Low-dose BU (10 mg/kg) was sufficient to allow human engraftment using 2 × 105 CD34+ cells, whereas higher doses (≥37.5 mg/kg) were toxic. NBSGW mice support human erythropoiesis in the bone marrow; however, murine macrophage depletion provided only minimal and transient increases in peripheral blood human erythrocytes. Our xenograft model is therefore useful in HSC gene therapy and genome-editing studies, especially for modeling in disorders, such as sickle cell disease, where access to HSCs is limited.

STEM-24. THERAPEUTIC APPROACH OF STEM CELL CARRYING RETROVIRAL REPLICATING VECTORS IN HUMAN GLIOMA MODEL

Abstract Toca 511, an improved retroviral replicating vectors (RRVs) expressing a codon-optimized cytosine deaminase, has shown highly promising evidence of therapeutic benefit in preclinical and clinical studies for gene therapy of glioma. In the present study, we engineered human mesenchymal stem cells (MSC) as tumor-homing cellular carriers that produce and release RRV, and evaluated the effect of this mode of virus delivery on the time course of intratumoral RRV dissemination. Human MSC isolates were engineered to produce RRV (MSC-RRV). Cytotoxicity assays confirmed efficient prodrug activator function in U-87 glioma cells transduced with vectors produced from MSC-RRV. To evaluate intratumoral migration activity and tumor-homing migration activity in vivo, individual MSC isolates were injected either directly into human glioma xenografts, or into the contralateral brain hemisphere. MSC-RRV isolates showing the highest levels of intratumoral migration activity were selected, and the efficiency of intratumoral dissemination and tumoricidal activity achieved by these isolates was compared against injection of RRV virus preparations in subcutaneous glioma models. Compared to virus injection, MSC-RRV achieved 1.6x-higher levels of tumor transduction (p< 0.05), and 2x-reduced tumor growth (p=0.018) at earlier time points. In short-term survival studies using lower doses of MSC-RRV cells vs. RRV virus injected 14 days post-establishment of intracranial U-87 gliomas, a small but statistically significant prolongation of median survival was seen with intracranial tumors treated with MSC-RRV as compared to RRV (26 vs. 20 days, p< 0.05) after only a single cycle of 5-FC prodrug. Thus, MSC can be employed as mobile tumor-homing RRV-producer cells, which release RRV as they migrate to tumor foci in vivo and actively penetrate into individual tumor masses, resulting in more rapid tumor transduction and earlier therapeutic efficacy, which may be advantageous particularly for multi-focal and metastatic disease. This study was funded by the California Institute for Regenerative Medicine (TR2-01791).

PBI75 REVIEW OF ECONOMIC EVALUATIONS FOR GENE THERAPIES:ARE WE READY FOR THE FUTURE?

High prices asked upfront for novel one-off gene therapies accompanied by uncertain long-term value claims raise concerns around reimbursement and affordability for Health Technology Assessments (HTAs) and payers. Economic evaluations (EEs) are commonly used to inform these reimbursement decisions. This study aimed to examine economic evaluation studies of Gene Therapies (GTs) and to provide an overview of the quality of the economic evidence available for use in decision making. We conducted a systematic review of peer reviewed literature to identify published EEs in English between 2007 and 2019. Data extracted from EEs included model, input and outcomes characteristics. Quality was assessed using the Consolidated Health Economic Evaluation Reporting Standards (CHEERS) checklist. A narrative analysis was performed in which we discussed methodology, assumptions and quality of EEs, as well as use and implications in decision-making. The systematic review yielded seven EEs of gene therapies. All EEs were cost-utility analyses and scored satisfactory to good on the CHEERS checklist. Five EEs modeled ‘traditional’ upfront payment and two an outcome-based payment scheme. Four studies concluded GTs were not cost-effective. Three EEs further explored different assumptions around long-term survival, alternative payment schemes (annuity-based payment, pay for performance) and their effect on the incremental cost-effectiveness ratio using extensive subgroup analyses or scenario’s. EEs with scenario analyses were considered most informative for decision making, providing insights in the effect of assumptions and feasibility alternative approaches. Although the quality of an EE may be considered good, their informativeness for HTA and payers and policy makers can be limited. We therefore recommend including scenario analyses in gene therapy EEs, simulating different effectiveness assumptions, payment scheme’s or survival extrapolation methods. With the introduction of new therapies comes the need to explore use and appropriateness of our evidence generation tools, including EEs, to inform our decisions.

Toward a Scalable Purification Protocol of GaLV-TR-Pseudotyped Lentiviral Vectors

Lentiviral vectors (LV) that are used in research and development as well as in clinical trials are in majority vesicular stomatitis virus G glycoprotein (VSVg) pseudotyped. The predominance of this pseudotype choice for clinical gene therapy studies is largely due to a lack of purification schemes for pseudotypes other than VSVg. In this study, we report for the first time the development of a new downstream process protocol allowing high-yield production of stable and infectious gibbon ape leukemia virus (GaLV)-TR-LV particles. We identified critical conditions in tangential flow filtration (TFF) and chromatographic steps for preserving the infectivity/functionality of LV during purification. This was carried out by identifying for each step, the critical parameters affecting LV infectivity, including pH, salinity, presence of stabilizers, temperature, and by defining the optimal order of these steps. A three-step process was developed for GaLV-TR-LV purification consisting of one TFF and two chromatographic steps (ion-exchange chromatography and size exclusion chromatography) permitting recoveries of >27% of infectious particles. With this process, purified GaLV-pseudotyped LV enabled the transduction of 70% human CD34+ cells in the presence of the Vectofusin-1 peptide, whereas in the same conditions nonpurified vector transduced only 9% of the cells (multiplicity of infection 20). Our protocol will allow for the first time the purification of GaLV-TR-LV that are biologically active, stable, and with sufficient recovery in the perspective of preclinical studies and clinical applications. Obviously, further optimizations are required to improve final vector yields.

Pilot Study of CSL200 Gene Therapy in Adults With Severe Sickle Sell Disease

Pilot Study of CSL200 Gene Therapy in Adults With Severe Sickle Sell Disease

Current Status on Clinical Development of Adeno-Associated Virus-Mediated Liver-Directed Gene Therapy for Inborn Errors of Metabolism.

Inborn errors of metabolism (IEM) are disorders affecting human biochemical pathways and represent attractive targets for gene therapy because of their severity, high overall prevalence, lack of effective treatments, and possibility of early diagnosis through newborn screening. The liver is a central organ involved in several metabolic reactions and is a favorite target for gene therapy in many IEM. Adeno-associated virus (AAV) vectors have emerged in the last years as the preferred vectors for in vivo gene delivery. Gene replacement strategies are aimed either at correcting liver disease or providing a source for production and secretion of the lacking enzyme for cross-correction of other tissues. A number of preclinical studies have been conducted in the last years and, for several diseases, gene therapy has reached the clinical stage, with a growing number of ongoing clinical trials. Moreover, recent applications of genome editing to the field of inherited metabolic diseases have further expanded potential therapeutic possibilities. This review describes relevant clinical gene therapy studies for IEM with particular attention to current obstacles and drawbacks.

Infectivity Assessment of Recombinant Adeno-Associated Virus and Wild-Type Adeno-Associated Virus Exposed to Various Diluents and Environmental Conditions.

Recombinant adeno-associated virus (rAAV) is a promising gene delivery vehicle that has been approved as a gene therapy drug for some genetic disorders, and is being evaluated in clinical trials. To further promote clinical research under the Food and Drug Administration Investigational New Drug application, the stability of rAAV must be assessed under various conditions. However, there is scant data concerning the stability of a variety of rAAV serotypes. We hypothesized that the difference of capsid structure causes differences in stability. To investigate this hypothesis, rAAV serotypes (rAAV1, rAAV2, rAAV8, and rAAV9) were exposed to diluents and various environmental conditions, including ultraviolet (UV) irradiation, 0.1 M sodium hydroxide (NaOH), 0.06% sodium hypochlorite (NaClO), tap water, and 70% ethanol (EtOH). The changes of the infectivity of the treated samples were assessed by transduction in HeLaRC32 cells as a criterion of stability. The infectivity between recombinant and wild-type AAV (wtAAV2) was also analyzed. The activity of all rAAV serotypes was weakened by UV irradiation and NaOH and NaClO exposure. Treatment for 10 days with tap water or 70% EtOH did not appreciably inactivate rAAV1, rAAV8, and rAAV9, but did affect the activity of rAAV2. Furthermore, the infectivity of rAAV2 did not surpass wtAAV2 infectivity. The results will be important for clinical studies for gene therapy using rAAV.

Advances and challenges for hemophilia gene therapy.

Hemophilia is an X-linked inherited bleeding disorder, resulting from defects in the F8 (hemophilia A) or F9 (hemophilia B) genes. Persons with hemophilia have bleeding episodes into the soft tissues and joints, which are treated with self-infusion of factor VIII or IX concentrates. Hemophilia provides an attractive target for gene therapy studies, due to the monogenic nature of these disorders and easily measurable endpoints (factor levels and bleed rates). All successful, pre-clinical and clinical studies to date have utilized recombinant adeno-associated viral (AAV) vectors for factor VIII or IX hepatocyte transduction. Recent clinical data have presented normalization of factor levels in some patients with improvements in bleed rate and quality of life. The main toxicity seen within these studies has been early transient elevation in liver enzymes, with variable effect on transgene expression. Although long-term data are awaited, durable expression has been seen within the hemophilia dog model with no late-toxicity or oncogenesis. There are a number of phase III studies currently recruiting; however, there may be some limitations in translating these data to clinical practice, due to inclusion/exclusion criteria. AAV-based gene therapy is one of a number of novel approaches for treatment of hemophilia with other gene therapy (in vivo and ex vivo) and non-replacement therapies progressing through clinical trials. Availability of these high-cost novel therapeutics will require evolution of both clinical and financial healthcare services to allow equitable personalization of care for persons with hemophilia.

Gene therapy for refractory angina and cell therapy for heart failure: experience of a Brazilian research group.

Cell therapy has shown impressive effects in experimental cardiomyopathy models. To a lesser extent, gene therapy has also been studied. In both cases, translation to clinical therapy has been disappointing. This paper is intended to describe the experience and achievements of a multicenter working group located in Porto Alegre, southern Brazil, in experimental and translational research projects for cell-based and gene therapy methods in the treatment of dilated and ischemic cardiomyopathies. The results of preclinical and clinical studies showed that bone marrow mononuclear stem cells indeed have an effect in improving myocardial perfusion and contractile function, but the overall results are poorly translated to the clinical level. Gene therapy studies with direct myocardial injections of naked VEGF 165 plasmid showed improvement in myocardial perfusion and function in animal models. A randomized clinical trial found that this method is safe and improved myocardial perfusion, but the benefits disappeared after 1 year. An animal experiment associating VEGF 165 with angiopoietin was undertaken in mini pigs to extend the durability of that therapy. In conclusion, our efforts to better understand the mechanisms and functions of gene and cell-based therapies in cardiology resulted in significant findings and propose a future look at cell-free therapeutic approaches.

Inducible cardiac-specific overexpression of cyclooxygenase-2 (COX-2) confers resistance to ischemia/reperfusion injury.

The role of cyclooxygenase-2 (COX-2) in cardiovascular biology remains controversial. Although COX-2 has been reported to mediate the protective actions of late preconditioning, other studies show that it is also an important mediator of inflammation, toxic shock, and apoptosis, resulting in significant dysfunction and injury in several tissues. To determine whether increased myocardial COX-2, in itself, is protective, cardiac-specific, inducible (Tet-off) COX-2 transgenic (iCOX-2 TG) mice were generated by crossbreeding α-MyHC-tTA transgenic mice (tetracycline transactivator [tTA]) with CMV/TRE-COX-2 transgenic mice. Three months after COX-2 induction, mice were subjected to a 30-min coronary occlusion and 24h of reperfusion. Three different lines (L5, L7, and L8) of iCOX-2 TG mice were studied; in all three lines, infarct size was markedly reduced compared with WT mice: L5 TG/TG 23.4 ± 5.8 vs. WT/WT 48.5 ± 6.1% of risk region; L7 TG/TG 23.2 ± 6.2 vs. WT/WT 53.3 ± 3.6%; and L8 TG/TG 23.5 ± 2.8 vs. WT/WT 52.7 ± 4.6% (P < 0.05 for each). COX-2 inhibition with NS-398 completely abolished the cardioprotection provided by COX-2 overexpression. This study for the first time utilizes an inducible cardiac-specific COX-2 overexpression system to examine the role of this enzyme in ischemia/reperfusion injury in vivo. We demonstrate that induced cardiac-specific overexpression of COX-2 exerts a potent cardioprotective effect against myocardial infarction in mice, and that chronic COX-2 overexpression is not associated with any apparent deleterious effects. We also show that PGE2 levels are upregulated in COX-2 overexpressing cardiac tissue, confirming increased enzyme activity. Finally, we have developed a valuable genetic tool to further our understanding of the role of COX-2 in ischemia/reperfusion injury and other settings. The concept that COX-2 is chronically protective has important therapeutic implications for studies of long-term gene therapy aimed at increasing myocardial COX-2 content as well as other COX-2- based strategies.

Abstract 4772: Evaluation of immune response following ultrasound targeted gene therapy in a murine model of prostate cancer

Abstract Human Adenoviruses (hAd) have been broadly used as gene delivery tool in preclinical and clinical studies of cancer gene therapy. The main challenges associated with systemic delivery of hAds are their high immunogenicity and host-specificity. Adenoviruses can elicit strong innate and acquired immune responses that reduce therapeutic gene transfer and expression in malignant sites. Murine cells generally lack some of the receptors necessary for hAd adhesion and internalization making them not permissive to infection and replication and limiting translational studies using murine models. We have developed a gene transfer method, which uses lipid-encapsulated perfluorocarbon microbubbles (MBs) and ultrasound (US) to protect the hAds from the immune system and to deliver them to a targeted tissue, bypassing the necessity for specific receptors. The expression of the Ad-receptors (CAR, Integrins αvβ3 and αvβ5), transduction efficiency of Ad-GFP, and GFP transgene expression were measured in vitro in murine (TRAMP-C2) and human (DU145) prostate cancer cells lines by flow cytometry and immunofluorescence. The activation of the innate and acquired immune system following the injection of Ad-GFP/MBs complexes was evaluated in vivo in C57BL/6 mice healthy or bearing a TRAMP-C2 syngeneic tumor graft. ELISA was used to quantify the inflammatory cytokines TNF-α, IL-6 and measure Neutralizing Antibodies. ELISpot assay was performed to measure the frequency of DBP-specific CD8+ T cells secreting INF-γ. We showed in vitro that murine TRAMP-C2 cells display an expression pattern of Ad-receptors comparable to human DU145 prostate cancer cells. We also demonstrated that both murine and human cells showed a dose dependent increase in the percentage of cells transduced by Ad-GFP at 24 hours. Additionally, we showed that TRAMP-C2 cells efficiently express the GFP transgene at 48 and 72 hours post transduction. To assess in vivo if our gene transfer method could effectively protect the Ads form both the innate and adaptive immunity, we injected C57BL/6 mice with the hAds-GFP/MBs complexes +/-US. Notably we did not observe activation of either innate (secretion of TNF- and IL-6 cytokines) or acquired immunity (neutralizing antibodies and presence of adenovirus-specific CD8+ T cells producing INF-γ). Our data provides evidence that the TRAMP-C2 syngeneic model of prostate cancer is a suitable system to study in immunocompetent mice the capability of the MBs/US to protect the Adenoviruses from the immune system while delivering them to a targeted site bypassing the requirement of specific receptors. Citation Format: Flavia De Carlo, Elliot T. Varney, Pier Paolo Claudio, Candace M. Howard. Evaluation of immune response following ultrasound targeted gene therapy in a murine model of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4772.

Pompe disease gene therapy: neural manifestations require consideration of CNS directed therapy.

Pompe disease is a neuromuscular disease caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase leading to lysosomal and cytoplasmic glycogen accumulation in neurons and striated muscle. In the decade since availability of first-generation enzyme replacement therapy (ERT) a better understanding of the clinical spectrum of disease has emerged. The most severe form of early onset disease is typically identified with symptoms in the first year of life, known as infantile-onset Pompe disease (IOPD). Infants are described at floppy babies with cardiac hypertrophy in the first few months of life. A milder form with late onset (LOPD) of symptoms is mostly free of cardiac involvement with slower rate of progression. Glycogen accumulation in the CNS and skeletal muscle is observed in both IOPD and LOPD. In both circumstances, multi-system disease (principally motoneuron and myopathy) leads to progressive weakness with associated respiratory and feeding difficulty. In IOPD the untreated natural history leads to cardiorespiratory failure and death in the first year of life. In the current era of ERT clinical outcomes are improved, yet, many patients have an incomplete response and a substantial unmet need remains. Since the neurological manifestations of the disease are not amenable to peripheral enzyme replacement, we set out to better understand the pathophysiology and potential for treatment of disease manifestations using adeno-associated virus (AAV)-mediated gene transfer, with the first clinical gene therapy studies initiated by our group in 2006. This review focuses on the preclinical studies and clinical study findings which are pertinent to the development of a comprehensive gene therapy strategy for both IOPD and LOPD. Given the advent of newborn screening, a significant focus of our recent work has been to establish the basis for repeat administration of AAV vectors to enhance neuromuscular therapeutic efficacy over the life span.

Company Profile: Aldevron Expands to Facilitate Emerging Therapies.

Company Profile: Aldevron Expands to Facilitate Emerging Therapies.

Enhanced Transduction of Macaca fascicularis Hematopoietic Cells with Chimeric Lentiviral Vectors.

Recent marketing approval for genetically engineered hematopoietic stem and T cells bears witness to the substantial improvements in lentiviral vectors over the last two decades, but evaluations of the long-term efficacy and toxicity of gene and cell therapy products will, nevertheless, require further studies in nonhuman primate models. Macaca fascicularis monkeys from Mauritius have a low genetic diversity and are particularly useful for reproducible drug testing. In particular, they have a genetically homogeneous class I major histocompatibility complex system that probably mitigates the variability of the response to simian immunodeficiency virus infection. However, the transduction of simian cells with human immunodeficiency virus type 1 (HIV-1)-derived vectors is inefficient due to capsid-specific restriction factors, such as the tripartite motif-containing protein tripartite motif 5α, which prevent infection with non-host-adapted retroviruses. This study introduced the modified capsid of the macaque-trophic HIV-1 clone MN4/LSQD into the packaging system and compared transduction efficiencies between hematopoietic cells transduced with this construct and cells transduced with HIV-1 NL4-3-derived packaging constructs. Capsid modification increased transduction efficiency in all hematopoietic cells tested (by factors of up to 10), including hematopoietic progenitor cells, repopulating cells, and T cells from Mauritian Macaca fascicularis, regardless of vector structure or purification method. The study also established culture conditions similar to those used in clinical practice for the efficient transduction of hematopoietic stem and progenitor CD34+ cells. These results suggest that the procedure is suitable for use in Mauritian Macaca fascicularis, which can therefore be used as a model in preclinical studies for hematopoietic gene and cell therapy.

Antibodies Tip the Balance Towards an HIV Cure

Long-term drug therapy for HIV-1 infection can prevent both disease progression and virus transmission, but treatment does not eradicate the virus. A new gene therapy study (Immunity 2019;50:567-575.e5) using human antibodies shows significant promise for a functional cure in a non-human primate model.

Clyde E. Keeler: The Rodless Mouse and the Early Days of Retinal Genetic Research

To report the lifetime activities and accomplishments of Clyde E. Keeler (1900-1994), a pioneer in the study of retinal genetics. Retrospective review. Assessment of published and unpublished biographical material. Nearly a century ago, Keeler discovered an inherited abnormality in the mouse that causes the absence of rod photoreceptors and is the mouse counterpart of 1 type of human retinitis pigmentosa. In 1923, Keeler serendipitously discovered the so-called rodless mouse, which is now known to be the result of a mutation in the PDEGB gene. The historical name for the mouse strain is rd. This same defect was reported in human patients with retinitis pigmentosa in 1993. Keeler's mouse model is still used in gene therapy studies seeking to cure retinitis pigmentosa.

Adenovirus-mediated Gene Therapy for Allergy

Allergy poses a heavy health burden in modern society. Other than symptom-relieving medications, the only available treatment approach is allergen-specific immunotherapy, which in spite of offering a potential cure, requires a long treatment duration with multiple doses of allergen administration and carries a risk of anaphylaxis. Gene therapy has shown advantages in experimental studies for treatment of tumors, genetic diseases, chronic infections, and allergy. To date, adenovirus has been the most extensively used gene transfer vector, and offers high efficiency and safety. Here, we review studies of adenovirus-mediated gene therapy targeting different steps in the development of allergic diseases. Adenovirus-mediated gene therapy might be a promising add-on therapy for allergy treatment.

Pre-existing antibodies to candidate gene therapy vectors (adeno-associated vector serotypes) in domestic cats.

Adeno-associated virus (AAV) vectors represent promising candidates for gene therapy; however, pre-existing neutralizing antibodies (NAb) may reduce AAV vector delivery efficiency. In this study, the presence of AAV NAb was investigated in cats, which serve as a larger and outbred animal model for the prediction of gene therapy outcomes in humans but also in cats.Serum/plasma samples from 230 client-owned Swiss cats and 20 specified pathogen-free cats were investigated for NAb to AAV1, AAV2, AAV5, AAV6, AAV7, AAV8 and AAV9 using in vitro transduction inhibition and a beta-galactosidase assay. NAb to all tested AAV serotypes were found. Of the client-owned cats, 53% had NAb to one or more of the AAV serotypes. NAb (≥1:10) were found at frequencies of 5% (AAV6) to 28% (AAV7). The highest titers were found against AAV7 (≥1:160). The NAb prevalence to AAV2, AAV7 and AAV9 differed geographically. Regarding titers ≥1:10 against single AAV serotypes, age, breed and sex of the cats were not associated with the NAb prevalence. Cats with titers ≥1:20 against AAV2 and titers ≥1:40 against AAV7 were significantly younger than cats with low/no titers, and purebred cats were significantly more likely than non-purebred cats to have NAb to AAV2 (≥1:40). Additionally, regarding NAb to all AAV combined, female cats were significantly more likely than male cats to have NAb titers ≥1:40. Preliminary data using AAV-DJ indicated that less pre-existing NAb to the hybrid AAV-DJ can be expected compared to the wild-type AAV serotypes. AAV NAb will need to be taken into account for future in vivo gene therapy studies in cats.

Glatiramer Acetate (Copaxone) is a Promising Gene Delivery Vector.

Glatiramer acetate (GA) is the active substance of Teva's Copaxone drug, which contains random polypeptides used to treat multiple sclerosis. Glatiramer acetate was originally developed to emulate human myelin basic protein, which contains four different residues [alanine (A), glutamic acid (E), tyrosine (T), and lysine (K)]. We found thatGA can complex, condense, and transfect plasmid DNA. Mixing the positively charged GA and the negatively charged genetic material in correct proportions produced small, stable, and highly positively charged nanoparticles. This simple GA-pDNA formulation produced high levels of transfection efficiency with low toxicity in HeLa and A549 cells (lung and cervical cancer cells). Additionally, we studied and compared the nanoparticle properties, gene expression, and cytotoxicity of K100-pDNA (high-molecular-weight polylysine) and K9-pDNA (low-molecular-weight polylysine) nanoparticles to those of GA-pDNA nanoparticles. We also studied the effect of calcium, which was previously reported to reduce the size and enhance gene expression resulting from similar polyelectrolyte complexes. Adding calcium did not reduce particle size, nor improve the transfection efficiency of GA-pDNA nanoparticles as it did for polylysine-pDNA nanoparticles. GA-pDNA nanoparticles may be prepared by mixing a genetic payload with approved GA therapeutics (e.g., Copaxone), thus offering intriguing possibilities for translational gene therapy studies.