Target Cells For Gene Therapy Research Articles

Usefulness of antibody-drug conjugate as preconditioning for hematopoietic stem cell-targeted gene therapy in wild-type and Fabry disease mouse models

BackgroundFabry disease (FD) is characterized by deficient activity of α-galactosidase A (GLA). Consequently, globotriaosylceramide (Gb3) accumulates in various organs, causing cardiac, renal, and cerebrovascular damage. Gene therapies for FD have been investigated in humans. Strong conditioning is required for hematopoietic stem cell-targeted gene therapy (HSC-GT). However, strong conditioning leads to various side effects and should be avoided. In this study, we tested antibody-based conditioning for HSC-GT in wild-type and FD model mice. MethodsAfter preconditioning with an antibody-drug conjugate, HSC-GT using a lentiviral vector was performed in wild-type and Fabry model mice. In the wild-type experiment, the EGFP gene was introduced into HSCs and transplanted into preconditioned mice, and donor chimerism and EGFP expression were analyzed. In the FD mouse model, the GLA gene was introduced into HSCs and transplanted into preconditioned Fabry mice. GLA activity and Gb3 accumulation in the organs were analyzed. ResultsIn the wild-type mouse experiment, when anti-CD45 antibody-drug conjugate was used, the percentage of donor cells at 6 months was 64.5%, and 69.6% of engrafted donor peripheral blood expressed EGFP. When anti-CD117 antibody-drug conjugate and ATG were used, the percentage of donor cells at 6 months was 80.7%, and 73.4% of engrafted donor peripheral blood expressed EGFP. Although large variations in GLA activity among mice were observed in the FD mouse experiment for both preconditioning regimens, Gb3 was significantly reduced in many organs. ConclusionsAntibody-based preconditioning may be an alternative preconditioning strategy for HSC-GT for treating FD.

Hematopoietic Stem Cell-Targeted Gene Therapy Using Lentiviral Vectors

Hematopoietic Stem Cell-Targeted Gene Therapy Using Lentiviral Vectors

Characterization of PDL1 enhanced siRNA/albumin liposome for effective therapeutic function in lung cancer.

The applications of liposomes are limited due to poor structural stability and short drug circulation time. This study aims to build an albumin-based liposomal delivery system to provide strategies for tumor specificity, efficient gene delivery and effective release of albumin liposomes. In this study, siRNA loaded PDL1-targeted albumin liposome was constructed for the treatment of lung cancer and its function was evaluated. Physical parameters such as particle size, potential and infrared spectrum were detected and microscopic morphology was observed by electron microscopy to detect the binding and uptake capacity of albumin liposome with cells. The optimal preparation process and binding ratio of PDL1-targeted albumin liposome/siRNA complex were determined. The constructed siRNA loaded PDL1-targeted albumin liposomes has low toxicity, high loading rate and tumor cell targeted gene therapy ability. Moreover, it increased T cell activation and down-regulated siRNA expression, effectively realizing the inhibition of lung cancer cells. The results showed that the PDL1-targeted albumin liposome could be used as a high efficient delivery vector of siRNA, and was a high efficient and safe nano vector for tumor targeted gene therapy.

A guide in lentiviral vector production for hard-to-transfect cells, using cardiac-derived c-kit expressing cells as a model system

Gene therapy revolves around modifying genetic makeup by inserting foreign nucleic acids into targeted cells via gene delivery methods to treat a particular disease. While the genes targeted play a key role in gene therapy, the gene delivery system used is also of utmost importance as it determines the success of gene therapy. As primary cells and stem cells are often the target cells for gene therapy in clinical trials, the delivery system would need to be robust, and viral-based entries such as lentiviral vectors work best at transporting the transgene into the cells. However, even within lentiviral vectors, several parameters can affect the functionality of the delivery system. Using cardiac-derived c-kit expressing cells (CCs) as a model system, this study aims to optimize lentiviral production by investigating various experimental factors such as the generation of the lentiviral system, concentration method, and type of selection marker. Our findings showed that the 2nd generation system with pCMV-dR8.2 dvpr as the packaging plasmid produced a 7.3-fold higher yield of lentiviral production compared to psPAX2. Concentrating the virus with ultracentrifuge produced a higher viral titer at greater than 5 × 105 infectious unit values/ml (IFU/ml). And lastly, the minimum inhibitory concentration (MIC) of puromycin selection marker was 10 μg/mL and 7 μg/mL for HEK293T and CCs, demonstrating the suitability of antibiotic selection for all cell types. This encouraging data can be extrapolated and applied to other difficult-to-transfect cells, such as different types of stem cells or primary cells.

AAV9-mediated Schwann cell-targeted gene therapy rescues a model of demyelinating neuropathy

Mutations in the GJB1 gene, encoding the gap junction (GJ) protein connexin32 (Cx32), cause X-linked Charcot-Marie-Tooth disease (CMT1X), an inherited demyelinating neuropathy. We developed a gene therapy approach for CMT1X using an AAV9 vector to deliver the GJB1/Cx32 gene under the myelin protein zero (Mpz) promoter for targeted expression in Schwann cells. Lumbar intrathecal injection of the AAV9-Mpz.GJB1 resulted in widespread biodistribution in the peripheral nervous system including lumbar roots, sciatic and femoral nerves, as well as in Cx32 expression in the paranodal non-compact myelin areas of myelinated fibers. A pre-, as well as post-onset treatment trial in Gjb1-null mice, demonstrated improved motor performance and sciatic nerve conduction velocities along with improved myelination and reduced inflammation in peripheral nerve tissues. Blood biomarker levels were also significantly ameliorated in treated mice. This study provides evidence that a clinically translatable AAV9-mediated gene therapy approach targeting Schwann cells could potentially treat CMT1X.

Mobilization of Auto-Stem Cells in the Thalassemia Major and Relationship with Iron Overload

Objectives: The hematopoietic stem cells transplantation (HSCT) is the only curable treatment in current for thalassemia major (TM).The quantity of auto stem cell of TM patients are very important for rescue the patients in case of failure of HSCT, and auto stem cells of TM will be used as target cells for gene therapy (GT) in the near future. Iron overload (IOL) can damage the hematopoiesis of TM. How to collect the auto stem cells in TM patients? How about the affects of iron overload in the mobilization and collection of auto stem cell? The aim of this study was to analyze quantity of auto stem cells in the TM and the affects of iron overload (IOL). Methods: We retrospectively analyzed a total of 134 patients and 25 normal donors undergoing PBSCs collection between January 2012 and December 2019. Patients with serum ferritin levels over 1,000 ng/mL and with a history of red blood cell transfusions prior to stem cell collection were defined as a group having transfusion-associated iron overload (IOL). In total one hundred and thirty-four patients and twenty-five donors were subcutaneous administrated G-CSF (Granulocyte Colony Stimulating Factor) 10 mg/kg/d Injection for 5 days, PBSCs was collected using a large volume leukapheresis (LVL) procedure. The data of WBC, MNC and CD34+ in product were analyzed by SPSS 20 software. Results: In 134 patients, median age is 8.60 range: (1-17),and 25 Normal donors, median age is 7.5 range: (1-18). The results demonstrated that cells either in patients with thalassemia or normal donors were effectively mobilized by G-CSF (10 mg / kg/d),as these children showed a marked increase in white blood cells and MNC cells in peripheral blood, reaching a peak in 4 to 5 days after the injection of G- CSF alone. Comparison of the MNC [(6.7±3.5) ×10⁸/kg vs (7.2±4.77) ×10⁸/kg] and CD34+cells [(10.29±4.5)×10⁶/kg vs (10.9±6.8)×10⁶/kg] in collected product in TM patients and normal donors groups revealed no significant difference. There was no significant difference between different Ferritin level (Mild, Intermediate and Severe) on WBC, MNC, and CD34+ in each group at the time point studied. But there was significant difference on MNC number (Normal liver iron (7.73±2.69)×10⁸/kg, Mild liver iron overload (7.66±4.64)×10⁸/kg, Intermediate (6.18± 2.84) ×10⁸/kg compared to Severe liver iron (4.45±3.34)×10⁸/kg, P=0.039. Significant difference was also can be seen in number of MNC of product in patients with intermediate cardiac iron overload compared to normal cardiac iron overload and mild cardiac iron overload (2.76±0.97) ×10⁶/kg vs(6.86±3.39) ×10⁶/kg and (6.88±3.72)×10⁶/kg, P=0.030. There was a statistically significant difference positive correlation between median age (P=0.011) and MNC/Kg (P=0.030). Conclusion:The quantity of mobilization of PBSC in TM patients had no significant difference compare to normal donor. The MNC in product was statistically decreased in intermediate cardiac iron overload and severe liver iron overload (IOL) subgroup. It indicated that iron overload (IOL) in organs may negatively relate to proliferation and mobilization of stem cells in TM. Further quality study of PBSC such as stem cell culture and ability of proliferation should be more evaluated. The sufficient quantity and high quality auto stem cell from TM patient can be more used in the future. Disclosures No relevant conflicts of interest to declare.

Efficient engraftment of genetically modified cells is necessary to ameliorate central nervous system involvement of murine model of mucopolysaccharidosis type II by hematopoietic stem cell targeted gene therapy

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disease (LSD) caused by a deficiency of the iduronate-2-sulfatase (IDS) that catabolizes glycosaminoglycans (GAGs). Abnormal accumulations of GAGs in somatic cells lead to various manifestations including central nervous system (CNS) disease. Enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT) are the currently available therapy for MPS II, but both therapies fail to improve CNS manifestations. We previously showed that hematopoietic stem cell targeted gene therapy (HSC-GT) with lethal irradiation improved CNS involvement in a murine model of MPS II which lacks the gene coding for IDS. However, the strong preconditioning, with lethal irradiation, would cause a high rate of morbidity and mortality. Therefore, we tested milder preconditioning procedures with either low dose irradiation or low dose irradiation plus an anti c-kit monoclonal antibody (ACK2) to assess CNS effects in mice with MPS II after HSC-GT. Mice from all the HSC-GT groups displayed super-physiological levels of IDS enzyme activity and robust reduction of abnormally accumulated GAGs to the wild type mice levels in peripheral organs. However, only the mice treated with lethal irradiation showed significant cognitive function improvement as well as IDS elevation and GAG reduction in the brain.These results suggest that an efficient engraftment of genetically modified cells for HSC-GT requires strong preconditioning to ameliorate CNS involvement in cases with MPS II.

Hematopoietic Stem Cell-Targeted Neonatal Gene Therapy with a Clinically Applicable Lentiviral Vector Corrects Osteopetrosis in oc/oc Mice

Infantile malignant osteopetrosis (IMO) is an autosomal recessive disorder characterized by nonfunctional osteoclasts. Approximately 50% of the patients have mutations in the TCIRG1 gene, encoding for a subunit of the osteoclast proton pump. Gene therapy represents a potential alternative treatment to allogeneic stem cell transplantation for IMO. The oc/oc mouse is a model of IMO characterized by a 1,500 bp deletion in the TCIRG1 gene, severe osteopetrosis, and a life span of only 3 weeks. Here we show that the osteopetrotic phenotype in oc/oc mice can be reversed by hematopoietic stem cell-targeted gene therapy with a clinically applicable lentiviral vector expressing a wild-type form of human TCIRG1 under the mammalian promoter elongation factor 1α short (EFS-hT). oc/oc c-kit+ fetal liver cells transduced with EFS-hT were transplanted into sublethally irradiated oc/oc mice by temporal vein injection 1 day after birth. A total of 9 of 12 mice survived long term (19-25 weeks) with evidence of tooth eruption, uncharacteristic of oc/oc mice. Splenocytes were harvested 19-25 weeks after transplantation and differentiated into osteoclasts on bone slices to assess resorption and on plastic to assess TCIRG1 protein expression. Vector-corrected osteoclasts showed human TCIRG1 expression by Western blot. CTX-I release relative to that mediated by oc/oc-derived osteoclasts increased 8-239-fold. Resorption pits on bone slices were observed for osteoclasts derived from 7/9 surviving transplanted oc/oc mice. Histopathology of the bones of surviving animals showed varying degrees of rescued phenotype, the majority with almost full correction. The average vector copy number per cell in the bone marrow was 1.8 ± 0.5. Overall, 75% of transplanted mice exhibited long-term survival and marked reversal of the osteopetrotic bone phenotype. These findings represent a significant step toward the clinical application of gene therapy for IMO.

Gene therapy for lysosomal storage diseases and peroxisomal diseases.

Gene therapies for lysosomal storage diseases (LSD) and peroxisomal diseases (PD) are rapidly advancing. Most LSDs and PDs are characterized by brain involvement, prompting the development of therapies targeting the brain. There are two types of gene therapy for brain involvement in LSD and PD, i.e., the direct transfer of a therapeutic gene into brain cells and hematopoietic stem cell-targeted gene therapy. The rationale for the latter approach is that brain microglia are derived from hematopoietic cells. Thus, gene-corrected hematopoietic cells migrate into the brain and differentiate into microglial cells. These gene-corrected microglial cells correct the metabolic defects associated with LSD and reduce inflammation in PD and LSD, leading to a clinical benefit. Gene editing technology has recently been applied in this area and a trial focused on LSD is currently ongoing. Although these approaches are still under investigation, very encouraging results have been obtained. This review provides an overview of recently developed gene therapies for various LSDs and PDs, including the results of clinical trials, with an emphasis on the benefits of this approach for these diseases.

Present Molecular Limitations of ON-Bipolar Cell Targeted Gene Therapy.

Recent studies have demonstrated the safety and efficacy of ocular gene therapy based on adeno-associated viral vectors (AAVs). Accordingly, a surge in promising new gene therapies is entering clinical trials, including the first optogenetic therapy for vision restoration. To date, optogenetic therapies for vision restoration target either the retinal ganglion cells (GCs) or presynaptic ON-bipolar cells (OBCs). Initiating light responses at the level of the OBCs has significant advantages over optogenetic activation of GCs. For example, important neural circuitries in the inner retina, which shape the receptive fields of GCs, remain intact when activating the OBCs. Current drawbacks of AAV-mediated gene therapies targeting OBCs include (1) a low transduction efficiency, (2) off-target expression in unwanted cell populations, and (3) a poor performance in human tissue compared to the murine retina. Here, we examined side-by-side the performance of three state-of-the art AAV capsid variants, AAV7m8, AAVBP2, and AAV7m8(Y444F) in combination with the 4xGRM6-SV40 promoter construct in the healthy and degenerated mouse retina and in human post-mortem retinal explants. We find that (1) the 4xGRM6-SV40 promoter is not OBC specific, (2) that all AAV variants possess broad cellular transduction patterns, with differences between the transduction patterns of capsid variants AAVBP2 and AAV7m8 and, most importantly, (3) that all vectors target OBCs in healthy tissue but not in the degenerated rd1 mouse model, potentially limiting the possibilities for an OBC-targeted optogenetic therapy for vision restoration in the blind.

Cell Compatibility of an Eposimal Vector Mediated by the Characteristic Motifs of Matrix Attachment Regions.

The characteristic sequence of β-interferon matrix attachment regions (MARs) can mediate transgene expression via episomal vectors in Chinese hamster ovary (CHO) cells. However, the host cells were from hamster ovaries, which are not suitable target cells for gene therapy. In this study, we aimed to evaluate the suitability of 12 different human cell lines as target cells for gene therapy. We transfected the cells with episomal vectors and obtained colonies stably expressing the vector products after G418 screening. Therefore the stably transfected cells were split into two and further cultured either in the presence or the absence of G418. Flow cytometry was used to observe the positive rate of cell transfection and level of green fluorescent protein (GFP) expression. Plasmid rescue assays, fluorescence in situ hybridization (FISH), and fluorescence quantitative polymerase chain reaction (PCR) were used to investigate the presence and gene copy numbers of plasmid in mammalian cells. The results showed that transfection efficiency and transgene expression levels in A375, Eca-109, and Changliver cells were high. In contrast, transgene silencing was observed in BJ, HSF, and A431 cells, and low expression of the transgene was observed in the other six cell lines. In addition, the plasmid was present in the episomal state in A375, Eca-109, and Chang-liver cells with relatively low copy numbers even under nonselective conditions. Thus, our results provide the first evidence showing transgene expression of an episomal vector mediated by the characteristic motifs of MARs for maintenance of the longterm stability of episomes in different types of cells.

Identification and Characterization of a Distinct, Evolutionarily Conserved HSC Phenotype Associated with and Predicting Multi-Lineage Engraftment

Current autologous stem cell transplantation and gene therapy strategies are limited by the inability to identify a true hematopoietic stem cell (HSC) propulation that reliably predicts engraftment. The ability to enrich and target a specific HSC pool and predict engraftment levels of the gene-modified cells would be a major advance, reducing manufacturing costs and off-target effects. Here we describe a distinct HSC phenotype, conserved between humans and nonhuman primates (NHP) which overcomes this limitation.We used our NHP stem cell transplantation and gene therapy model to study the engraftment potential of phenotypically distinct hematopoietic stem and progenitor cell (HSPC) subpopulations. We were able to identify an exclusive HSPC subpopulation capable of multi-lineage engraftment (Figure 1A and 1B). This HSPC subpopulation (denoted "I") accounts for ~3-5% of the entire CD34+ cell population in primed bone marrow, reducing the number of cells targeted for cell and gene therapy approaches by 20 to 30-fold. For autologous transplants, only 300-400K HSPCs/kg body weight were required to achieve rapid neutrophil and plateletet recovery within 9-10 and 19-20 days, respectively. Stable 30-35% gene-marking was obeserved in all blood lineages including T cell, B cell, NK cells, granulocyte, monocytes/macrophages, erythrocytes and platelets. Complete reconstitution of the bone marrow compartment was achieved within <100 days, with 31-33% gene-marking in HSCs as well as downstream progenitors from all lineages.Most importantly, and in support of the above described engraftment studies, the use of this HSPC population I phenotype allowed us to establish a reliable flow cytometry based analysis to assess and predict engraftment after HSC transplantion. This method predicted the onset of neutrophil and platelet recovery (Figure 1C and 1D). This assay was also able to predict the onset of NHP hematopoietic recovery for transplanted HSCs from different stem cell sources including steady state (unprimed) bone marrow, as well as gene-modified/transduced and ex vivo expanded HSCs. From this data, we were able to define the minimum cell number of HSCs/kg body weight required for sustained multi-lineage long-term engraftment with recovery of all blood cell lineages in the NHP transplant setting as 110,000 cells/kg. Importantly, we demonstrated that this cell population and phenotype is conserved between NHP and human hematopoiesis (Radtke et al. 2016, submitted).In summary, we identified a distinct and evolutionarily conserved HSC phenotype that is associated with multi-lineage engraftment in nonhuman primates and also reliably predicted the engraftment kinetics after transplant. This will greatly facilitate evaluation and "potency" of autolgous stem cell products especially after stem cell expansion or gene therapy. In addition, it will allow for an improved enrichment of the target cells for gene therapy and gene editing protocols and thus likely improve efficacy and safety. [Display omitted] DisclosuresAdair:Rocket Pharmaceuticals: Consultancy, Equity Ownership. Kiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Research Funding.

Aerosol delivery of folate-decorated hyperbranched polyspermine complexes to suppress lung tumorigenesis via Akt signaling pathway

Lung cancer has been a leading cause of cancer mortality worldwide and aerosol-mediated gene therapy endows numerous advantages compared to other traditional modalities. Here, we reported a folic acid (FA)-modified hyperbranched polyspermine (HPSPE) with prominent biocompatibility for lung cancer cell targeted gene therapy. FA was decorated to the HPSPE via an amidation reaction and the physicochemical properties of nanoplexes formed with DNA were characterized. Gel electrophoresis study elucidated that the designed polymer was capable to condense DNA and protect it from degradation by DNase I. Cell viability and transfection efficiency assay in vitro and in vivo indicated its increased transfection performance with lower toxicity. Furthermore, reduced tumor numbers and down-regulation of Akt1 protein after aerosol treatment containing FA-HPSPE/shAkt1 complexes proved its therapeutic potential for lung cancer suppression. Results obtained in this study suggested that FA-HPSPE with highly biocompatibility and targeting capability while forming complexes with shAkt1 and administrated through noninvasive aerosol could be prospective for inhibiting lung tumorigenesis.

Brain endothelial cell-targeted gene therapy of neurovascular disorders.

Neurovascular disorders are difficult to treat due to the blood–brain barrier (BBB), which prevents delivery of most drugs from the blood into the brain. Gene therapy can potentially overcome this barrier, but classical vectors are neither efficient nor specific enough to provide long‐lasting treatment or avoid off‐target effects. In this issue of EMBO Molecular Medicine , Korbelin et al (2016) describe an engineered adeno‐associated virus (AAV) with exceptional tropism for the brain that restores a normal phenotype in a mouse model of incontinentia pigmenti (IP), a severe human genetic disorder of brain vasculature.

基因修饰的骨髓间充质干细胞移植治疗帕金森病的研究进展

帕金森病进展缓慢，以神经退化为特征，主要病因是选择性中脑黑质多巴胺能神经元的丧失和纹状体多巴胺含量的显著减少。传统疗法有药物治疗和外科治疗，而近来较理想是基因治疗和干细胞移植治疗，包括胚胎干细胞、神经干细胞和间充质干细胞等。骨髓间充质干细胞作为体内一种多功能干细胞，具有成为基因治疗靶细胞的显著优势，并已被普遍用于人类疾病的研究。然而，对帕金森病进行基因治疗的目的基因选择具有多样性。因此，本文主要针对近十年基因治疗帕金森病的目的基因的选择相关研究进展作一综述。 Parkinson’s disease (PD) is a neurodegenerative disease characterized by progressive degeneration of nigrostriatal dopaminergic (Dopamine, DA) neurons and reduced concentration of striatal dopamine. Traditional therapies for Parkinson’s disease are medication and surgery, while gene therapy and stem-cell therapy have been considered as much more ideal methods recently, such as embryonic stem cells, neural stem cells and mesenchymal stem cells, etc. Bone mesenchymal stem cells as a kind of pluripotent stem cells in the body are widely used in the related human diseases for the superiority to be the target cells of gene therapy. However, choosing appropriate target genes for therapy of Parkinson’s disease has a great diversity, therefore, here I summarize the researches of the theraphy methods of Parkinson’s disease in recent 10 years.

Sheltering DNA in self-organizing, protein-only nano-shells as artificial viruses for gene delivery

By recruiting functional domains supporting DNA condensation, cell binding, internalization, endosomal escape and nuclear transport, modular single-chain polypeptides can be tailored to associate with cargo DNA for cell-targeted gene therapy. Recently, an emerging architectonic principle at the nanoscale has permitted tagging protein monomers for self-organization as protein-only nanoparticles. We have studied here the accommodation of plasmid DNA into protein nanoparticles assembled with the synergistic assistance of end terminal poly-arginines (R9) and poly-histidines (H6). Data indicate a virus-like organization of the complexes, in which a DNA core is surrounded by a solvent-exposed protein layer. This finding validates end-terminal cationic peptides as pleiotropic tags in protein building blocks for the mimicry of viral architecture in artificial viruses, representing a promising alternative to the conventional use of viruses and virus-like particles for nanomedicine and gene therapy. From the Clinical EditorFinding efficient gene delivery methods still represents a challenge and is one of the bottlenecks to the more widespread application of gene therapy. The findings presented in this paper validate the application of end-terminal cationic peptides as pleiotropic tags in protein building blocks for “viral architecture mimicking” in artificial viruses, representing a promising alternative to the use of viruses and virus-like particles for gene delivery.

Plerixafor Single Agent for Autologous Stem Cells Mobilization and Collection in Adult Thalassemic Patients: Towards the Assessment of the Suitable Hematopoietic Stem Cell Source for Gene Therapy of Beta-Thalassemia.

AbstractAbstract 586Gene therapy of inherited blood diseases requires harvest of hematopoietic stem cells (HSCs) from patients and autologous transplantation of genetically modified cells. In order to achieve correction of the disease, high number of HSCs and previous conditioning of the host bone marrow (BM) are necessary. In the clinical application of gene therapy for thalassemic patients the choice of the HSC source is a crucial issue. On one side, the minimal target dose poses a challenge for the use of steady state BM since reinfusion of high numbers of beta globin gene modified CD34+ cells is probably necessary to gain sufficient correction of the genetic defect in order to achieve transfusion independency; on the other side, the disease related features and complications of thalassemic patients (i.e. splenomegaly and thrombophilia) dictate caution in the use of G-CSF as mobilizing agent.In April 2011 a clinical protocol exploring the use of Plerixafor (AMD3100) as single agent was started (“Plerixafor mobilized stem cells as source for gene therapy of beta-thalassemia”, acronym AMD-THAL, EudraCT2011-000973-30). Aims of the trial were to explore the ability of Plerixafor in inducing safe and effective stem cells mobilization in adult patients affected by beta-thalassemia, to characterize stem/progenitor cells mobilized from the BM and peripheral blood of treated subjects and to achieve gene transfer efficiency of mobilized CD34+ cells at a level comparable to that obtained using steady state BM.Four patients (01, 02, 03 and 04) were enrolled and already mobilized to date (August 2012). All patients are affected by transfusion dependent beta-thalassemia and aged 28 (01), 41 (02), 39 (03), 33 (04). Two are splenectomized (02 and 03); all subjects are regularly iron chelated with adequate organ function. Administration of Plerixafor subcutaneously as single agent and at the single dose of 0.24 mg/kg resulted in mobilization of CD34+ cells/mcl with a peak of 78 cells at 9 hrs (01), 70 cells at 7 hrs (02) and 69 cells at 8 hrs (03); suboptimal mobilization was observed in patient 04 (peak 18 at 8 hrs). Patient 03 received a second dose at 0.40 mg/kg 24 hrs after the first dose and underwent a second leucoapheretic procedure. Harvest by leukoapharesis resulted in procurement of the following CD34+ cells/kg: 1.84 × 106 (01) and 4.43 × 106 (02) with a unique leukoapheretic procedure, and 3.57 × 106 (03) with two leukoapheresis. No apheresis was performed for patient 04 because the minimum target of 20 CD34+ cells/mcl in peripheral blood was not reached. CD34+ cells selection through Clinimacs Miltenyi resulted in the following yield: 1.2 × 106 CD34+ cells/Kg, 65% recovery (01), 2.66 × 106 CD34+ cells/Kg, 60% recovery (02), 1.78 × 106 CD34+ cells/Kg, 50% recovery (03). No severe adverse event occurred. Recorded side effects were: grade 3 hypotension related to the apheretic procedure (01), mild grade 1 facial disestesia (02 and 04) and hyperleukocytosis (02: WBC from 13.6 to 42.6 × 103/mcl). In addition, steady state and Plerixafor primed BM aspirates were performed to analyze any modification in CD34+ concentration in the BM following Plerixafor administration. In fact, Plerixafor administration resulted in enrichment of CD34+ cells concentration in the BM.Purified CD34+ cells from leukoapheresis of the 4 treated patients were analyzed for their biological and functional properties, subpopulations composition and expression profile. In vivo reconstitution potential and lymphomyeloid differentiation of CD34+ cells were tested following transplantation in NSG mice. Experiments are ongoing but preliminary results indicate that cells mobilized by Plerixafor have a primitive phenotype with a high reconstitution potential and are efficiently transduced with a lentiviral based vector, named GLOBE, encoding for the human beta-globin (Roselli et al., 2010), thus being a suitable source of target cells for gene therapy. Disclosures:No relevant conflicts of interest to declare.

Pseudotyped AAV Vector-Mediated Gene Transfer in a Human Fetal Trachea Xenograft Model: Implications for In Utero Gene Therapy for Cystic Fibrosis

BackgroundLung disease including airway infection and inflammation currently causes the majority of morbidities and mortalities associated with cystic fibrosis (CF), making the airway epithelium and the submucosal glands (SMG) novel target cells for gene therapy in CF. These target cells are relatively inaccessible to postnatal gene transfer limiting the success of gene therapy. Our previous work in a human-fetal trachea xenograft model suggests the potential benefit for treating CF in utero. In this study, we aim to validate adeno-associated virus serotype 2 (AAV2) gene transfer in a human fetal trachea xenograft model and to compare transduction efficiencies of pseudotyping AAV2 vectors in fetal xenografts and postnatal xenograft controls.Methodology/Principal FindingsHuman fetal trachea or postnatal bronchus controls were xenografted onto immunocompromised SCID mice for a four-week engraftment period. After injection of AAV2/2, 2/1, 2/5, 2/7 or 2/8 with a LacZ reporter into both types of xenografts, we analyzed for transgene expression in the respiratory epithelium and SMGs. At 1 month, transduction by AAV2/2 and AAV2/8 in respiratory epithelium and SMG cells was significantly greater than that of AAV2/1, 2/5, and 2/7 in xenograft tracheas. Efficiency in SMG transduction was significantly greater in AAV2/8 than AAV2/2. At 3 months, AAV2/2 and AAV2/8 transgene expression was >99% of respiratory epithelium and SMG. At 1 month, transduction efficiency of AAV2/2 and AAV2/8 was significantly less in adult postnatal bronchial xenografts than in fetal tracheal xenografts.Conclusions/SignificanceBased on the effectiveness of AAV vectors in SMG transduction, our findings suggest the potential utility of pseudotyped AAV vectors for treatment of cystic fibrosis. The human fetal trachea xenograft model may serve as an effective tool for further development of fetal gene therapy strategies for the in utero treatment of cystic fibrosis.

Helper-dependent Adenoviral Vectors are Superior In Vitro to First-generation Vectors for Endothelial Cell-targeted Gene Therapy

Arterial endothelial cells (EC) are attractive targets for gene therapy of atherosclerosis because they are accessible to hematogenous and catheter-based vector delivery and overlie atherosclerotic plaques. Vector-mediated expression-in EC-of proteins that mediate cholesterol transfer out of the artery wall and decrease inflammation could prevent and reverse atherosclerosis. However, clinical application of this strategy is limited by lack of a suitable gene-transfer vector. First-generation adenovirus (FGAd) is useful for EC gene transfer in proof-of-concept studies, but is unsuitable for atheroprotective human gene therapy because of limited duration of expression and proinflammatory effects. Moreover, others have reported detrimental effects of FGAd on critical aspects of EC physiology including proliferation, migration, and apoptosis. Here, we investigated whether helper-dependent adenovirus (HDAd) either alone or expressing an atheroprotective gene [apolipoprotein A-I (apoA-I)] could circumvent these limitations. In contrast to control FGAd, HDAd did not alter any of several critical EC physiologic functions (including proliferation, migration, apoptosis, metabolic activity, and nitric oxide (NO) production) and did not stimulate proinflammatory pathways [including expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and interleukin-6 (IL-6)]. Expression of apoA-I by HDAd reduced EC VCAM-1 expression. HDAd is a promising vector and apoA-I is a promising gene for atheroprotective human gene therapy delivered via EC.

Synthetic Peptides As Non-Viral DNA Vectors

The use of multiple peptide motifs to provide effective gene delivery holds great promise as an elegant, non-immunogenic approach to gene therapy. The molecular understanding of cell and viral biology provides a strong foundation on which to pursue this objective. Synthetic peptides containing multiple lysines and/or arginines (occasionally ornithines) provide natural polycations for multivalent electrostatic binding of DNA, and for DNA compaction into particles suitable for gene delivery. These cationic peptides can incorporate additional functional motifs (e.g. for translocating DNA into the nucleus) and they can be linked by disulphide bonds to produce high molecular reducible polycations with superior properties for gene therapy. Many factors influence the size, surface charge and stability of peptide/DNA particles. For in vivo use, uncharged particles resistant to disruption by salt and protein, and targeted to tissue-specific membrane molecules, will be required. Entry into the cell is via one of the endocytic pathways, depending on particle size and (in principle) the target cell surface molecule. Peptide motifs for endocytic escape are based mainly on the anionic fusogenic peptide of influenza virus haemagglutinin and on histidine-rich peptides (where the buffering properties of the imidazole group cause osmotic swelling and probably rupture of endocytic vesicles). Once in the cytosol, translocation of DNA plasmids across the nuclear pore complex into the nucleus is a crucial step, because most target cells for gene therapy are either non-dividing or slowly dividing. Nuclear translocation can be achieved by classical nuclear localising motifs, or more simply by (Lys)16 and other cationic peptides.