Dao Pan

Real-Time Quantitative Polymerase Chain Reaction to Assess Gene Transfer

Accurate quantification of gene transfer (or gene correction) is a universal challenge in the field of gene therapy. In developing a clinical trial of lymphocyte gene therapy for Hunter syndrome (mucopolysaccharidosis type II), methods using Southern blot or automated DNA sequencing technology were employed, but found to be laborious and subject to considerable variation. As an alternative approach, we explored a real-time kinetic PCR assay appropriate to new instrumentation (PE Biosystems model 7700). A TaqMan probe was designed to hybridize directly across the exon 2-exon 3 junction of the iduronate-2-sulfatase transgene cDNA. In this assay system, cDNA from the retroviral vector L2SN generates a PCR product that is 84 nucleotides long and readily quantified by TaqMan probe binding and subsequent cleavage. Evaluation of this method demonstrated sensitivity over at least 5 logs with respect to the standard (vector plasmid pL2SN). There was no detectable signal from genomic DNA from nontransduced cells, thus indicating the specificity of this assay. The sample preparation method used to prepare specimens was a relatively simple cell lysis procedure, without DNA extraction, and represents a significant advancement over the more complex methods of DNA extraction that are typically used for such assays. This specific assay, and comparison to previous methods, illustrates the utility of a new method that is readily generalized to many gene therapy studies, and that has the potential to be extended to measure gene expression by means of quantitative RT-PCR.

Retroviral transduction and expansion of peripheral blood lymphocytes for the treatment of mucopolysaccharidosis type II, Hunter's syndrome.

BACKGROUND: Gene therapy using autologous peripheral blood lymphocytes (PBLs) has been used to produce adenosine deaminase with which to treat patients with severe combined immunodeficiency. Patients with mucopolysaccharidosis type II (MPS II) lack iduronate‐2‐sulfatase (IDS), and serial PBL gene therapy may benefit these patients.

Closed Hollow-fiber Bioreactor: a New Approach to Retroviral Vector Production

The ability to obtain high‐titer and large quantities of retroviral vector production in a ‘closed’ system would have profound implications in clinical and experimental gene therapy.

Retroviral vector design studies toward hematopoietic stem cell gene therapy for mucopolysaccharidosis type I

To optimize a gene transfer system for hematopoietic stem cell gene therapy of patients with mucopolysaccharidosis (MPS) type I, 10 retroviral vectors were constructed to express the human α-L-iduronidase (IDUA) cDNA. These vectors were designed to evaluate the potential effects of specific promoters, the addition of selectable markers, and the use of multiple promoters versus an internal ribosome entry site for expression of IDUA and selectable maker genes. The effect of vector design was investigated in primary patient fibroblasts (FMPS) or murine fibroblast cell lines; while overall comparison of transgene expression was determined in patients’ peripheral blood lymphocytes (PBLMPS) and CD34+ progenitors (PBPCMPS). We observed that the human PGK promoter introduced the highest IDUA activity per 1% relative transgene frequency in FMPS. Use of the same promoter to separately regulate both the therapeutic gene and a drug-resistance gene resulted in decreased expression of the unselected gene. Co-selection using bicistronic vectors not only increased the number of transductants, but also elevated transgene expression under selective pressure in transgene-positive progenitors. Bicistronic vector LP1CD overcame down-regulation and practically introduced the highest IDUA level in unselected PBLMPS and an intermediate level in PBPCMPS. These studies provide a better understanding of factors contributing to efficient gene expression in hematopoietic cells.

Craniofacial abnormalities in a murine knock-out model of mucopolysaccharidosis I H: a computed tomography and anatomic study.

The genetic mucopolysaccharidoses are a group of lysosomal storage diseases in which mucopolysaccharides (glycosaminoglycans) accumulate as the result of a malfunction or lack of a lysosomal degradation enzyme. There are currently seven known forms of mucopolysaccharidoses. Type I results from an enzymatic deficiency of alpha-l-iduronidase. There are three subtypes of mucopolysaccharidoses I that are commonly recognized: Hurler syndrome, Hurler-Scheie syndrome, and Scheie syndrome. Of the three subtypes, Hurler syndrome has the most severe clinical picture. Craniofacial anomalies and cognitive impairment are some of the more pronounced features of Hurler syndrome. Hurler syndrome has been described in cats, dogs, mice, and human beings and is inherited as an autosomal recessive trait. The biochemical nature of the disease is preserved across species lines. Clinically, the disease has similar effects in human beings and animals. It has been difficult to reverse the phenotype of the disease even with replacement of the defective alpha-l-iduronidase enzyme. The purpose of this study is to characterize the cranio-facial differences in the murine knock-out model of Hurler syndrome objectively. Twenty-three measurements were taken from computed tomographic scans in a coronal and sagittal plane on 24 black C57/B6 knock-out Hurler syndrome mice. The seven statistically significant measurements are width of the cervical canal, height of the foramen magnum, width between the external auditory canals, width of the skull base at the mandibular condyles, midocular distance, spread of the mandibular condyles, and width of the zygoma at the maxilla. This information now provides researchers with objective data from living Hurler syndrome-affected mice that will allow them to follow therapies directed at improving craniofacial outcomes for any therapy over time.

Improved gene transfer and normalized enzyme levels in primitive hematopoietic progenitors from patients with mucopolysaccharidosis type I using a bioreactor.

One of the major barriers to the clinical application of hematopoietic stem cell (HSC) gene therapy has been relatively low gene transfer efficiency. Other inadequacies of current transduction protocols are related to their multi‐step procedures, e.g., using tissue‐culture flasks, roller bottles or gas‐permeable bags for clinical application.

Combined Ultrafiltration-Transduction in a Hollow-Fiber Bioreactor Facilitates Retrovirus-Mediated Gene Transfer into Peripheral Blood Lymphocytes from Patients with Mucopolysaccharidosis Type II

The process of growing and transducing large quantities of human primary peripheral blood lymphocytes (PBLs) with high gene transfer efficiency continues to be one of the major challenges for clinical and experimental gene therapy. Toward developing a clinical trial of lymphocyte gene therapy for mucopolysaccharidosis type II (i.e., Hunter syndrome), we investigated a novel method that exploited the innate capability of a hollow-fiber bioreactor system to filter large quantities of vector supernatant and facilitate transduction. An aliquot (5 x 10(7)) of PBL apheresis product was precultured in a gas-permeable culture bag or a bioreactor, and then transduced with a retroviral vector L2SN containing the iduronate-2-sulfatase (IDS) and neomycin resistance genes. We observed that the total number of PBLs could be expanded up to 187-fold, yielding up to 10(10) cells at the end of a 7-day culture period. The multiplicity of infection could be increased (up to 20-fold) by ultrafiltrating a large volume of vector supernatant through the semipermeable membrane of this system. A high level of transduction efficiency (up to 57%) was achieved, resulting in IDS enzyme activity as high as 1250 U/mg/hr in transduced PBL(MPS) 15 days after transduction. This level was markedly increased from that of nontransduced cells (<3 U/mg/hr) and was even greater than that of normal PBLs (mean, 809; n = 10). After 12 days of G418 selection, PBL(MPS) transductants exhibited a proviral IDS enzyme level approximately threefold higher than that in normal PBLs. These results indicated that the hollow-fiber bioreactor could be used to culture and transduce human primary PBLs in clinically useful quantities with relatively high gene transfer efficiency and transgene expression.

Retroviral transduction of peripheral blood leukocytes in a hollow- fiber bioreactor

BACKGROUND: Peripheral blood white cells (leukocytes) (PBLs) have been used as effective targets for genetic manipulation by transduction with retroviruses in open systems. A semi‐closed hollow‐fiber bioreactor was tested for culturing and transducing lymphocytes. STUDY DESIGN AND METHODS: PBLs were isolated from five healthy donors, and 5 × 10(7) cells were cultured in hollow‐fiber bioreactors for 4 days after stimulation with anti‐CD3 in medium containing 200 units per mL of recombinant interleukin 2. Transduction with retroviral vector containing the gene for iduronate‐2‐sulfatase and G 418 resistance, L2SN, was performed daily on Days 4, 5, 6, and 7, and the cells were expanded for an additional 3 days. RESULTS: PBLs from three donors were harvested from the bioreactor after transduction and expansion, and 4.5 × 10(9), 7.0 × 10(9), and 2.9 × 10(9) cells were recovered, representing 90‐, 136‐, and 58‐fold expansions. The transduction frequency of L2SN was 10, 5, and 1 percent, respectively. For additional expansion of PBLs, in two cases the bioreactor was reinoculated with 5 × 10(7) cells, which were expanded again for 16 and 8 days, respectively, yielding 1.4 × 10(9) and 3.1 × 10(9) cells, which reflected an additional 28‐ and 62‐fold expansion of cells. PBLs from two other donors were transduced and expanded in the bioreactor, and then 0.8 mg per mL of G 418 was added to the medium in an attempt to enrich the transduced population. Although 2.5 and 10 percent of the cells were transduced, cell death and absence of expansion in the presence of G 418 resulted in final cell lots with viabilities of only 4 and 8 percent. In all cases, the harvested cells tested negative in bacterial and fungal cultures. CONCLUSION: Hollow‐fiber bioreactors are an efficient and effective system for the retroviral transduction and expansion of PBLs for clinical gene therapy.