High-efficiency transduction and long-term gene expression with a murine stem cell retroviral vector encoding the green fluorescent protein in human marrow stromal cells.

Bone marrow stromal cells (MSCs) are unique mesenchymal cells that have been utilized as vehicles for the delivery of therapeutic proteins in gene therapy protocols. However, there are several unresolved issues regarding their potential therapeutic applications. These include low transduction efficiency, attenuation of transgene expression, and the technical problems associated with drug-based selection markers. To address these issues, we have developed a transduction protocol that yields high-level gene transfer into human MSCs, employing a murine stem cell virus-based bicistronic vector containing the green fluorescent protein (GFP) gene as a selectable marker. Transduction of MSCs plated at low density for 6 hr per day for 3 days with high-titer viral supernatant resulted in a gene transfer efficiency of 80+/-6% (n = 10) as measured by GFP fluorescence. Neither centrifugation nor phosphate depletion increased transduction efficiency. Assessment of amphotropic receptor (Pit-2) expression by RT-PCR demonstrated that all MSCs expressing the receptor were successfully transduced. Cell cycle distribution profiles measured by propidium iodide staining showed no correlation with the susceptibility of MSCs to transduction by the retroviral vector. Human MSCs sequentially transduced with an adenoviral vector encoding the ecotropic receptor and ecotropic retroviral vector encoding GFP demonstrated that all MSCs are susceptible to retroviral transduction. We further showed that both genes of bicistronic vector are expressed for at least 6 months in vitro and that transgene expression did not affect the growth or osteogenic differentiation potential of MSCs. Future studies will be directed toward the development of gene therapy protocols employing this strategy.

Targeted integration of a recombinant globin gene adeno-associated viral vector into human chromosome 19.

Transfer of a globin gene into stem cells along with the regulatory elements required to achieve high level expression in maturing erythroid cells would provide effective gene therapy for Cooley's Anemia. We have explored the use of recombinant adeno-associated viral (rAAV) vectors for this purpose. A vector designated rHS32A gamma*3'RE that contains regulatory elements from the locus control and flanking regions, integrates as a stable head-to-tail concatamer in erythroleukemia cells at a high multiplicity of infection and exhibits high level, regulated gamma globin gene expression. Inducible expression of the non-structural Rep proteins of wild-type AAV in HeLa cells transduced with rAAV vectors does not increase overall integration frequency, but targeted integration of rHS32A gamma*'3'RE into human chromosome 19 was documented.

Use of bolus intraperitoneal iron dextran in continuous ambulatory peritoneal dialysis or continuous cyclic peritoneal dialysis patients receiving recombinant human erythropoietin.

Impaired erythropoiesis in continuous ambulatory peritoneal dialysis (CAPD) or continuous cyclic peritoneal dialysis (CCPD) patients receiving recombinant human erythropoietin (rHuEPO) is most often secondary to iron deficiency, either as a result of poor intestinal absorption or failure to take oral supplements as prescribed. The inconvenience of giving intravenous (i.v.) iron dextran (ID) to CAPD/CCPD patients precluded its use in this population. We therefore examined the efficacy of bolus intraperitoneal (i.p.) iron dextran (1000 mg) on erythropoiesis in a pilot study of 14 CAPD/CCPD patients. The patients ranged in age from 23-81 years, and all had iron deficiency (transferrin saturation 6%-23%; mean: 15.2% +/- 1.34%). Of the 14 patients studied, 13 were receiving rHuEPO. Pre-treatment hematocrit (Hct) ranged from 21%-38% (mean: 30.2% +/- 1.37%). After infusion of 2 L Dianeal (Baxter Healthcare Corp., Deerfield, Illinois, U.S.A.), 500 mg of undiluted ID was administered directly into the Tenckhoff catheter and subsequently flushed with 30 mm3 normal saline. The peritoneal dialysis (PD) exchange containing ID then dwelled for a period not < 6 hours before standard PD resumed. A second 500 mg dose ID was given to each patient by the same protocol 3-86 days later (mean: 14 days). No complications were seen. No patient complained of abdominal pain or other subjective symptoms during infusion or during the dwell. Repeat iron studies done 1-7 months post ID (mean: 2.8 months) showed a 1.1-fold to 4.9-fold increase (mean: 1.4-fold) in mean iron levels (40.4 +/- 3.9 mg/dL versus 57.5 +/- 5.5 mg/dL, p = 0.036); a 1.1-fold to 5.2-fold increase (mean: 1.6-fold) in mean transferrin saturation (15.2% +/- 1.3% versus 24.5% +/- 2.6%, p = 0.008); a 1.01-fold to 1.60-fold increase (mean: 1.12-fold) in mean Hct (30.2% +/- 1.37% versus 33.8% +/- 1.5%; p = 0.042). The mean dose of rHuEPO was statistically unchanged (170.0 +/- 47.4 U/kg body weight versus 178.8 +/- 49.6 U/kg body weight per week; p = 0.841). Peritoneal equilibration test (PET) score 1-4 months post ID (mean: 2 months) was 0.778 +/- 0.02 compared with a PET score at baseline of 0.767 +/- 0.03 (p = 0.734). No significant delta was observed in blood urea nitrogen (BUN) or creatinine values. We conclude that use of bolus i.p. ID is safe, effective, and convenient, and demonstrates no short-term negative effect on peritoneal membrane integrity. Long-term effects have yet to be determined.

Use of bolus intraperitoneal aminoglycosides for treating peritonitis in end-stage renal disease patients receiving continuous ambulatory peritoneal dialysis and continuous cycling peritoneal dialysis.

Peritonitis is the most common complication of peritoneal dialysis (PD) and accounts for the most drop-out from PD among patients with end-stage renal disease (ESRD). Antibiotic protocols to treat peritonitis recommend initial treatment for both gram-positive and gram-negative infections, pending culture results. Current literature also suggests comparable therapeutic success using bolus (single-dose) parenteral aminoglycosides to treat systemic gram-negative infections compared to conventional divided-dose aminoglycosides. The single bolus dose antibiotic regimen has the potential for reduced nephrotoxicity and ototoxicity. We therefore hypothesized that intermittent bolus dose intraperitoneal (i.p.) aminoglycosides may be superior to conventional continuous dose i.p. aminoglycosides in treating dialysis-associated peritonitis, and have fewer side effects. Six patients with clinical peritonitis were treated with single, bolus-dose, i.p. gentamicin or tobramycin (5 mg/kg ideal body weight), range 250-440 mg (mean: 355 +/- 68.25 mg) at the start of therapy. No patient grew gram-negative organisms; aminoglycosides were therefore not repeated. Three patients used four continuous ambulatory peritoneal dialysis (CAPD) exchanges per day; three patients used six nightly continuous cycling peritoneal dialysis (CCPD) exchanges with a daytime dwell. Mean PD aminoglycoside clearance was 6.75 +/- 2.27 mm3/min; mean urinary aminoglycoside clearance was 5.75 +/- 1.14 mm3/min. Mean blood aminoglycoside elimination t1/2 was 29.27 +/- 3.55 hours, with a mean blood level of 3.18 +/- 1.45 micrograms/mL 72 hours after initial therapy, and 1.52 +/- 0.81 micrograms/mL 96 hours after initial therapy. Peritoneal equilibration test (PET) scores before and after aminoglycoside administration (performed a mean of 4.6 months apart) were 0.672 +/- 0.097 and 0.705 +/- 0.092 respectively (p = 0.321). Comparative audiograms using pure-air tone conduction with frequencies from 250-12,000 Hz were done within 24 hours of aminoglycosides and again when therapy was complete (mean: 17 days). No significant changes were seen. While efficacy of bolus versus conventional-use aminoglycosides could not be definitely established, the kinetics of bolus aminoglycosides suggests that therapeutic blood levels persist for 72-96 hours and that the risk for oto/vestibular toxicity is negligible. We conclude that use of bolus i.p. aminoglycosides is safe, achieves therapeutic blood levels for extended intervals, demonstrates no clinical oto/vestibular toxicity, is cost-effective, and is a convenient strategy for patients and nursing staff.

The use of stress by language-impaired children.

This investigation was conducted to identify the stressing patterns of language-impaired children in the early stages of language acquisition. Based on Wieman's (1976) work with children who were acquiring language normally, it had been expected that the subjects of this study would tend to stress the new information in two-word utterances. Only one of the five preschool language-impaired children used such a pattern. Three subjects tended to stress words in the final position and one of the subjects' preference was unclear. The results, although preliminary, provide support for the contention that language-impaired children may differ from normal children in their use of stress.

Evidence for development of distinction of voice onset time in a child with left-hemisphere lesion.

Following surgery for partial removal of the posterior left hemisphere at 5 mo., voice onset time was assessed to 9; 11 yr. Left-hemisphere language function associated with voicing appeared subsumed by the right hemisphere.

Gene transfer into hematopoietic cells.

Transfer of a gene into stem cells with subsequent lineage-specific gene expression is a desired goal with many potential therapeutic applications. Retroviral vectors developed from murine leukemia viruses reproducibly transfer genes into murine stem cells, but are inefficient at gene insertion into stem cells of larger animals or man. A growing knowledge of stem cell biology suggests that this inefficiency reflects the quiescent state of stem cells, even when incubated in the presence of multiple cytokines and low expression of the receptor for amphotropic retroviral vectors. Alternative vector systems are being explored in an effort to overcome these barriers to stem cell-targeted gene transfer. Our work has shown that recombinant adeno-associated virus vectors, which have the potential for transducing quiescent cells, transfer, express and integrate a globin gene linked to its normal regulatory elements in human erythroid cells, but only at very high multiplicities of infection. The integrated genome was stable and the encoded globin gene was expressed at levels equivalent to a normal globin gene.

High-level globin gene expression mediated by a recombinant adeno-associated virus genome that contains the 3' gamma globin gene regulatory element and integrates as tandem copies in erythroid cells.

Recombinant adeno-associated virus (rAAV) vectors are being evaluated for gene therapy applications. Using purified rAAV containing a mutationally marked globin gene (A(gamma)*) and sites 2, 3, and 4 from the locus control region (rHS432A(gamma)*), but lacking a drug-resistance gene, we investigated the relationship between multiplicity of infection (MOI), gene expression, and unselected genome integration in erythroid cells. Most primary erythroid progenitors were transduced as reflected by A(gamma)* mRNA in mature colonies but only at an MOI of greater than 5 x 10(7). Using immortalized erythroleukemia cells as a model, we found that fewer than one half of the colonies that contained the A(gamma)* transcript had an integrated, intact rHS432A(gamma)* genome. rHS432A(gamma)* integrated as a single copy with expression at approximately 50% the level of an endogenous gamma globin gene. A second vector, rHS32A(gamma)*3'RE, containing the regulatory element (RE) from 3' to the chromosomal A(gamma) globin gene, integrated as an intact, tandem head to tail concatamer with a median copy number of 6 with variable expression per copy ranging from approximately onefold to threefold that of an endogenous y globin gene. These results establish that purified rAAV can be used to achieve integration and functional expression of a globin gene in erythroid cells, but only when high MOIs are used.

Cloning and expression of murine sister of P-glycoprotein reveals a more discriminating transporter than MDR1/P-glycoprotein.

Sister of P-glycoprotein (SPGP), a novel murine cDNA and member of the ATP-binding cassette superfamily highly homologous to P-glycoprotein (Pgp), was cloned. Moreover, its genomic clone was isolated and localized to chromosome 2 by fluorescence in situ hybridization. SPGP was functionally evaluated relative to MDR1 after subcloning SPGP cDNA into a retroviral bicistronic vector capable of expressing both SPGP and the green fluorescent protein. LLC-PK1 and MDCKII cells were transduced with this retrovirus and SPGP-positive clones were isolated. Drug uptake and efflux was compared in cells ectopically expressing either SPGP or human MDR1. SPGP cells had decreased uptake of taurocholate and vinblastine compared with LLC-PK1 cells. Additional studies revealed that vinblastine efflux was accelerated by SPGP compared with LLC-PK1. Further comparison revealed that although MDR1 easily impaired uptake of vincristine, daunomycin, paclitaxel, and digoxin, SPGP had no effect on uptake of these drugs. However, further studies demonstrated that, like MDR1, SPGP effluxed calcein-acetoxymethyl ester (AM). Unlike MDR1, SPGP was incapable of effluxing rhodamine 123. Although cyclosporine A and reserpine blocked calcein-AM transport by MDR1, these drugs had either minimal or no effect, respectively, on blocking SPGP efflux of calcein-AM. In contrast, ditekiren, a linear hexapeptide, readily and preferentially inhibited SPGP efflux of calcein-AM. Further studies with three structural analogs of ditekiren revealed that one analog inhibited SPGP efflux of calcein-AM, although not as potently as ditekiren. These are the first studies to reveal that SPGP has distinct transport properties compared with MDR1.