Leukemia inhibitory factor improves survival of retroviral vector-infected hematopoietic stem cells in vitro, allowing efficient long-term expression of vector-encoded human adenosine deaminase in vivo.

Low recovery and poor retroviral vector infection efficiency of hematopoietic stem cells has hindered application of gene therapy for disease affecting blood-forming tissues. Developmental restriction (or death) of stem cells during ex vivo infection has contributed to these difficulties. In these studies we report that the cytokine leukemia inhibitory factor (LIF) directly or indirectly supported the survival of hematopoietic stem cells during culture of bone marrow with vector-producing fibroblasts, resulting in efficient recovery of stem cells able to compete for engraftment in irradiated recipient animals. The infection efficiency of hematopoietic stem cells recovered from these cultures was approximately 80%; and all recipients (20/20) of the LIF-treated marrow were stably engrafted with the progeny of provirus-bearing stem cells. Expression of vector-encoded human adenosine deaminase (hADA) was detected in all recipients at levels averaging 15-50% of endogenous murine ADA in all their hematolymphoid tissues. Survival of stem cells in untreated cultures was approximately 10% of that observed from LIF-treated cultures, resulting in poor engraftment of recipient animals with transplanted cells. The infection efficiency of the few stem cells recovered from untreated cultures, however, was high (approximately 80%), suggesting that LIF did not have an effect on infection efficiency per se, but acted at the level of stem cell survival. Consistent with the poor engraftment observed in the control animals, expression of vector-encoded ADA was only approximately 4-20% of the endogenous levels. These results support the postulated role of LIF as a regulator of hematopoiesis and suggest that cytokine stimulation can positively affect inefficient retroviral vector transduction in hematopoietic stem cells.

20p12.3 microdeletion predisposes to Wolff-Parkinson-White syndrome with variable neurocognitive deficits.

BACKGROUND: Wolff-Parkinson-White syndrome (WPW) is a bypass re-entrant tachycardia that results from an abnormal connection between the atria and ventricles. Mutations in PRKAG2 have been described in patients with familial WPW syndrome and hypertrophic cardiomyopathy. Based on the role of bone morphogenetic protein (BMP) signalling in the development of annulus fibrosus in mice, it has been proposed that BMP signalling through the type 1a receptor and other downstream components may play a role in pre-excitation. METHODS AND RESULTS: Using the array comparative genomic hybridisation (CGH), we identified five individuals with non-recurrent deletions of 20p12.3. Four of these individuals had WPW syndrome with variable dysmorphisms and neurocognitive delay. With the exception of one maternally inherited deletion, all occurred de novo, and the smallest of these harboured a single gene, BMP2. In two individuals with additional features of Alagille syndrome, deletion of both JAG1 and BMP2 were identified. Deletion of this region has not been described as a copy number variant in the Database of Genomic Variants and has not been identified in 13 321 individuals from other cohort examined by array CGH in our laboratory. CONCLUSIONS: Our findings demonstrate a novel genomic disorder characterised by deletion of BMP2 with variable cognitive deficits and dysmorphic features and show that individuals bearing microdeletions in 20p12.3 often present with WPW syndrome.

Amerindian ancestry in Argentina is associated with increased risk for systemic lupus erythematosus.

Previous studies have demonstrated that in admixed populations, West African ancestry is associated with an increased prevalence of systemic lupus erythematosus (SLE). In the current study, the effect of Amerindian ancestry in SLE was examined in an admixed population in Argentina. The Argentine population is predominantly European with approximately 20% Amerindian admixture, and a very small (<2%) contribution from West Africa. The results indicate that Amerindian admixture in this population is associated with a substantial increase in SLE susceptibility risk (Odds Ratio=7.94, P=0.00006). This difference was not due to known demographic factors, including site of collection, age and gender. In addition, there were trends towards significance for Amerindian ancestry influencing renal disease, age of onset and anti-SSA antibodies. These studies suggest that populations with Amerindian admixture, like those with West African admixture, should be considered in future studies to identify additional allelic variants that predispose to SLE.

Insights into lymphocyte development from X-linked immune deficiencies.

The genetic immune deficiencies have drawn the attention of physicians and immunologists for more than 40 years. The selectivity of these deficiencies brings into focus the contribution of the response of each arm of the immune system to specific pathogens. Recently, the genes underlying four X-linked defects in immune development in humans have been identified by either positional cloning or candidate-gene cloning techniques. Remarkably, these genetic defects reveal a microcosm of lymphocyte developmental controls involving cell-cell interactions, combinatorial cell surface receptor specificity and lineage-specific signal transduction.

A single strand conformation polymorphism study of CD40 ligand. Efficient mutation analysis and carrier detection for X-linked hyper IgM syndrome.

Mutations in the gene for CD40 ligand are responsible for the X-linked form of hyper IgM syndrome. However, no clinical or laboratory findings that reliably distinguish X-linked disease from other forms of hyper IgM syndrome have been reported, nor are there tests available that can be used to confidently provide carrier detection. To identify efficiently mutations in the gene for CD40 ligand, eight pairs of PCR primers that could be used to screen genomic DNA by single strand conformation polymorphism (SSCP) were designed. 11 different mutations were found in DNA from all 13 patients whose activated T cells failed to bind a recombinant CD40 construct. The exact nature of four of these mutations, a deletion and three splice defects, could not be determined by cDNA sequencing. In addition, SSCP analysis permitted rapid carrier detection in two families in whom the source of the mutation was most likely a male with gonadal chimerism who passed the disorder on to some but not all of his daughters. These studies document the utility of SSCP analysis for both mutation detection and carrier detection in X-linked hyper IgM syndrome.

Expression of human adenosine deaminase in murine hematopoietic cells.

Multiple replication-defective retrovirus vectors were tested for their ability to transfer and express human adenosine deaminase in vitro and in vivo in a mouse bone marrow transplantation model. High-titer virus production was obtained from vectors by using both a retrovirus long terminal repeat promoter and internal transcriptional units with human c-fos and herpes virus thymidine kinase promoters. After infection of primary murine bone marrow with one of these vectors, human adenosine deaminase was detected in 60 to 85% of spleen colony-forming units and in the blood of 14 of 14 syngeneic marrow transplant recipients. This system offers the opportunity to assess methods for increasing efficiency of gene transfer, for regulation of expression of foreign genes in hematopoietic progenitors, and for long-term measurement of the stability of expression in these cells.

Cell cycle-dependent expression and nucleolar localization of hCAP-H.

Condensin is a conserved 13S heteropentamer composed of two nonidentical structural maintenance of chromosome (SMC) family proteins, in Xenopus XCAP-C and XCAP-E, and three regulatory subunits, XCAP-D2, XCAP-G, and XCAP-H. Both biochemical and genetic analyses have demonstrated an essential role for the 13S condensin complex in mitotic chromosome condensation. Further, a potential requirement for condensin in completion of chromatid arm separation in early anaphase is demonstrated by the mutational phenotypes of the Drosophila homologues of XCAP-H, barren and XCAP-C, DmSMC4. In this study we have investigated the expression and subcellular distribution of hCAP-H, the human homolog of XCAP-H, in order to better understand its cellular functions. Transcription of hCAP-H was restricted to proliferating cells with highest expression during the G(2) phase of the cell cycle. In contrast, cellular hCAP-H protein levels were constant throughout the cell cycle. hCAP-H was found to be associated with mitotic chromosomes exhibiting a nonuniform but symmetric distribution along sister chromatids. The symmetry of hCAP-H association with sister chromatids suggests that there are sequence-dependent domains of condensin aggregation. During interphase hCAP-H, -C, and -E, have distinct punctate nucleolar localization, suggesting that condensin may associate with and modulate the conformation and function of rDNA. hCAP-H association with condensed chromatin was not observed in the early phase of chromosome condensation when histone H3 phosphorylation has already taken place. This finding is consistent with the hypothesis that histone H3 phosphorylation precedes condensin-mediated condensation.

Long-distance DD-PCR and cDNA microarrays.

Analysis of differential gene expression is a classic tool in experimental biology. Broadly applicable new methods to identify and quantitative differential mRNA profiles, such as long distance differential display PCR and cDNA microarrays, promise to greatly accelerate understanding of mechanisms of development, differentiation, and disease.

De novo deletions and duplications of 17q25.3 cause susceptibility to cardiovascular malformations.

BACKGROUND: Genomic disorders resulting from deletion or duplication of genomic segments are known to be an important cause of cardiovascular malformations (CVMs). In our previous study, we identified a unique individual with a de novo 17q25.3 deletion from a study of 714 individuals with CVM. METHODS: To understand the contribution of this locus to cardiac malformations, we reviewed the data on 60,000 samples submitted for array comparative genomic hybridization (CGH) studies to Medical Genetics Laboratories at Baylor College of Medicine, and ascertained seven individuals with segmental aneusomy of 17q25. We validated our findings by studying another individual with a de novo submicroscopic deletion of this region from Cytogenetics Laboratory at Cincinnati Children's Hospital. Using bioinformatic analyses including protein-protein interaction network, human tissue expression patterns, haploinsufficiency scores, and other annotation systems, including a training set of 251 genes known to be linked to human cardiac disease, we constructed a pathogenicity score for cardiac phenotype for each of the 57 genes within the terminal 2.0 Mb of 17q25.3. RESULTS: We found relatively high penetrance of cardiovascular defects (~60 %) with five deletions and three duplications, observed in eight unrelated individuals. Distinct cardiac phenotypes were present in four of these subjects with non-recurrent de novo deletions (range 0.08 Mb-1.4 Mb) in the subtelomeric region of 17q25.3. These included coarctation of the aorta (CoA), total anomalous pulmonary venous return (TAPVR), ventricular septal defect (VSD) and atrial septal defect (ASD). Amongst the three individuals with variable size duplications of this region, one had patent ductus arteriosus (PDA) at 8 months of age. CONCLUSION: The distinct cardiac lesions observed in the affected patients and the bioinformatics analyses suggest that multiple genes may be plausible drivers of the cardiac phenotype within this gene-rich critical interval of 17q25.3.

Stable RNA secondary structure in a retroviral vector insert terminates reverse transcriptase elongation in vitro but not in cultured cells.

We wished to test whether an RNA signal that causes termination of elongation by reverse transcriptase in vitro would affect retroviral vector function. A synthetic oligonucleotide containing a sequence capable of forming a very stable RNA secondary structure was subcloned into the retrovirus vector N2. The integration of this sequence into N2 causes termination of elongation by reverse transcriptase in vitro at the precise positions previously reported in a different sequence context. However, no premature termination of DNA synthesis was observed in the unintegrated DNA of vector transduced cells. Likewise, there was no deleterious effect of the sequence insert on vector titer. These results indicate that termination signals defined in in vitro systems cannot be used as predictors of in vivo function and suggest that viral proteins in addition to reverse transcriptase play an important role in transcript initiation and elongation.

Evaluation of lymphoid-specific enhancer addition or substitution in a basic retrovirus vector.

Two novel retroviral vectors bearing lymphoid-specific enhancers were tested for improved expression of human adenosine deaminase (hADA) in tissue culture cells and in mouse bone marrow transplant recipients. These vectors carried either an added human T-cell receptor alpha-chain enhancer (delta N2TADA) or a substitution of the Moloney long terminal repeat (LTR) enhancer with the murine immunoglobulin mu heavy-chain first intron enhancer (delta N2 mu ADA). Each vector was produced at a titer of approximately 10(6) infectious units/ml and efficiently transduced hADA into murine fibroblast and myeloma cells in culture. No quantitative difference in expression was observed between the enhancer modified vectors and the basic retrovirus vector (delta N2ADA). In addition, each vector efficiently conferred hADA expression in lymphoid, myeloid, and erythroid cells of long-term transplanted mice. The majority of the transduced-marrow recipients demonstrated expression of the human enzyme for 4-8 months with each of the three vectors.

In vivo marking of spontaneous or vaccine-induced fibrosarcomas in the domestic house cat, using an adenoviral vector containing a bifunctional fusion protein, GAL-TEK.

We evaluated the ability of a replication-deficient, recombinant adenoviral vector to transfer the bifunctional gene GAL-TEK, which expresses a marking/therapeutic gene product, to naturally occurring cat fibrosarcomas in situ. GAL-TEK contains an in-frame fusion of the bacterial LacZ gene for histochemical marking of tumors with beta-galactosidase (beta-Gal) and the HSV tk gene for enzyme-prodrug activation of the prodrug ganciclovir (GCV) to induce selective tumor cell killing. GAL-TEK bifunctional marking and cell killing activities were tested in vitro after adenoviral vector infection of HT1080 human fibrosarcoma cells. The tk activity of GAL-TEK is shown to be almost as potent as HSV tk to catalyze conversion of GCV to GCV nucleotides and promote selective cell killing. Using 8 cats with recurring 2.5-cm2 fibrosarcomas that either arose spontaneously or were induced by vaccine, we determined experimentally the administration routes and times required for optimum GAL-TEK gene transfer by beta-Gal histological staining and reverse transcriptase polymerase chain reaction to the multiple compartments of the growing fibrosarcomas consonant with minimizing collateral infection of neighboring tissues and other unwanted side effects.

Delivery of a secretable adenosine deaminase through microcapsules--a novel approach to somatic gene therapy.

Many current gene therapy protocols require genetic modification of autologous cells. An alternate approach is to use universal recombinant cell lines engineered to secrete in vivo the desired gene products. Enclosing these cells within immunoprotective devices before implantation would prevent rejection of the nonautologous donor cells. To overcome the limitation that not all therapeutic gene products are secreted, we now propose to fuse a signal sequence to the amino terminus of a nonsecreted protein such as human adenosine deaminase (ADA), thus directing the product into a secretory pathway for release from the cells. A fusion gene constructed between the cDNA of the beta-lactamase signal sequence and human ADA expressed a product after in vitro transcription and translation that was immunologically similar to the human protein. Mouse fibroblasts transfected with the fusion gene demonstrated secreted ADA activity that resembled the human cytosolic enzyme in its heat stability, pH optimum, KM, electrophoretic mobility, and immunologic reactivity. Hence, the secreted enzyme expressed from the fusion gene is antigenically and enzymatically similar to the authentic human form. When transfected mouse fibroblasts or myoblasts were enclosed in permselective alginate-poly-L-lysine alginate microcapsules, ADA activity was secreted from the microcapsules and the cells remained viable for over 5 months. Hence, a secretable and functional human ADA has been constructed that can be delivered from recombinant cells within immunoprotective capsules. The success of this strategy provides the prototype for engineering nonsecreted gene products for therapy via this novel method of somatic gene therapy.

A new family of murine retroviral vectors with extended multiple cloning sites for gene insertion.

Murine retroviral vectors with multiple unique cloning sites in the body and 3' long terminal repeat (LTR) are described. The various alterations to the vectors include changing the gag+ start codon (AUG) to a stop codon (UAA), a deletion of 468 bp from the envelope region, and an additional 387-bp deletion of the promoter and enhancer sequences from the 3' LTR. Multiple cloning sites in the body and 3' LTR facilitate double-copy vector construction. The hygromycin resistance and luciferase genes were subcloned into the body and 3' LTR to evaluate effects of vector modifications and effects of insert location (body vs. LTR and same orientation vs. reverse orientation with respect to the vector LTRs) on virus titer. The results indicate the modifications or insert position do not negatively influence potential vector titer and expression capacity. The described vectors have potentially useful characteristics for gene therapy studies.

Protection of primary human T cells from HIV infection by Trev: a transdominant fusion gene.

Gene therapy is one of several approaches that are being tested in the search for an effective anti-human immunodeficiency virus (HIV) treatment. In this strategy, a "protective" gene would be introduced into target cells, rendering them relatively resistance to the virus-induced cytopathicity. Tat and Rev are viral proteins essential for HIV gene expression. Tat increases viral gene transcription and Rev is responsible for the nuclear export of mRNA encoding structural viral proteins. A fusion protein (Trev) was constructed, joining Tat and Rev transdominant mutant gene sequences. Previously, we showed that Trev inhibits both Tat and Rev activities in Jurkat T cells. To determine whether Trev could inhibit HIV replication in primary cells, we transferred the trev gene to peripheral blood lymphocytes and challenged them with different HIV strains. Levels of HIV p24 antigen (Ag) were reduced 4- to 15-fold in cultures of Trev-CD4+ T cells infected with two HIV primary clinical isolates and were not detectable in cultures infected with HIV strains NL4-3 and SF2. In contrast, cultures of nontransduced CD4+ T cells infected with the same viruses had levels of HIV p24 Ag up to 10 ng/ml. Trev-transduced CD4+ T cells demonstrated increased survival following HIV challenge for the length of the experiments (30 days). We did not observe rapid emergence of Trev-resistant HIV in our cultures. Following HIV challenge, cell-associated Trev protein was increased, supporting the hypothesis that cells surviving Trev expression provided a cell survival advantage. This work showed that Trev was able to inhibit HIV replication in primary CD4+ T cells, and, therefore the trev gene could be a candidate for gene therapy against HIV.

Gene transfer of adenosine deaminase into primitive human hematopoietic progenitor cells.

The inherited deficiency in adenosine deaminase (ADA), which results in severe combined immunodeficiency, is generally regarded as an optimal model for the development of human somatic gene therapy. The ideal target for the correction of ADA deficiency and other lympho-hematopoietic disorders would be the hematopoietic stem cell. We have used a combination of recombinant human interleukins-3 and -6 to stimulate the proliferation of primitive human hematopoietic progenitor cells during a period of co-cultivation with irradiated cells producing high titers of an ADA-transducing retroviral vector packaged in amphotropic particles. In a series of nine experiments, an average of 83% of the clonogenic progenitors (CFU-E and CFU-GM) were found to have acquired the transferred sequence as determined by polymerase chain reaction analysis. In addition, in two experiments, 24-44% of the clonogenic progenitors derived from long-term myeloid cultures 9 weeks post-transduction were found to contain vector sequence. The latter cells are derived from so-called "long-term culture-initiating cells" (LTC-IC), which are primitive cells probably related to hematopoietic stem cells. Moreover, the transduced ADA enzyme was found to be expressed in both normal and ADA-deficient erythroid colonies, and in the nonadherent cells of long-term bone marrow culture for at least 2 weeks at levels that approximate the endogenous ADA levels of normal erythroid cells. These results indicate that the ADA coding sequence can efficiently be introduced by retroviral gene transfer into both committed and primitive human hematopoietic progenitor cells, and that this will result in adequate expression of the transduced enzyme in the progeny of committed hematopoietic progenitors.

Expression of human CD18 in murine granulocytes and improved efficiency for infection of deficient human lymphoblasts.

The CD18 gene encodes the beta 2-subunit of leukocyte integrins, and mutations in this gene cause extreme host susceptibility to bacterial and fungal infection. Because expression of CD18 is restricted to bone marrow-derived cells, this disorder is considered an excellent candidate for somatic gene therapy utilizing ex vivo infection of bone marrow stem cells. We have constructed a retroviral vector expressing CD18 with the Moloney murine leukemia virus (Mo-MLV) long terminal repeat (LTR) as the promoter, and high-titer ecotropic and amphotropic producer cell lines were isolated using the GP+E-86 and GP+envAM12 safe packaging cell lines. Infection of CD18-deficient lymphoblasts resulted both in expression of immunodetectable CD18 at 35-40% of normal levels on 55-60% of cells and in functional restoration of CD18-dependent aggregation. All of 16 mice transplanted with syngeneic bone marrow infected with the CD18 retrovirus expressed human CD18 on 17-36% of granulocytes at 2 weeks after transplantation, and expression was appropriately up-regulated in response to stimulation with zymosan-activated serum. This recombinant retrovirus should prove useful for further studies of somatic gene therapy for CD18 deficiency.

Basal ganglia calcifications in a case of biotinidase deficiency.

Biotinidase deficiency leads to a biotin-deficient state, with cardinal symptoms of ataxia, alopecia, and skin rash presenting in infancy. Previous reports of head CTs in patients with biotinidase deficiency did not note basal ganglia calcifications. We report the first case of biotinidase deficiency with basal ganglia calcifications. There were no symptoms referable to basal ganglia dysfunction.

A sensitive reporter cell line for HIV-1 tat activity, HIV-1 inhibitors, and T cell activation effects.

The production and characterization of a cell line for quantitative HIV-1 Tat function and T cell activation assays is described. 1G5 is a clonal cell line derived from Jurkat T cells stably transfected with a luciferase gene driven by an HIV-1 long terminal repeat (HIV-LTR). The 1G5 clone was selected for low basal luciferase activity, susceptibility to HIV infection, and high responsiveness to Tat and T cell activation signals. A 10 to 1000-fold increase in luciferase activity after transfection or infection with tat-expressing vectors or HIV was observed. Equivalent levels of expression were detected after stimulation with T cell mitogens. The characteristics of 1G5 make it a valuable reagent for studies of HIV infection, HIV regulatory agents, and other T cell or HIV-activating factors, and for screening potential anti-HIV therapeutic agents.

Mild phenotypic effects of a de novo deletion Xpter-->Xp22.3 and duplication 3pter-->3p23.

We report on a girl with a de novo monosomy Xpter-->Xp22.3 and trisomy 3pter-->3p23, normal development and stature, mildly affected phenotype, and learning disabilities with a low normal level of intelligence. Late replication studies using BudR demonstrated that the entire der(X) was inactive in 30% of cells. In 62% of cells the inactivation did not spread to the autosomal segment in the der(X). The normal X was inactivated in 8% of cells. Quantitative X-inactivation studies using the human androgen receptor locus assay (HAR) on peripheral leukocytes and buccal epithelial cells showed extreme skewing of methylation (90.4% of the paternal allele). The correlation of cytogenetic and molecular data suggest that the mild phenotype of the proposita is most likely due to preferential inactivation of the entire der(X), which seems to be of paternal origin.