Gene therapy for lysosomal storage disorders.

Deficiencies in one or several of the numerous degradative enzymes that reside in the lysosome often result in one of many clinically severe diseases, almost all of which have no currently available therapy. Although bone marrow transplantation, enzyme replacement and substrate inhibition therapies are being considered, gene therapy represents an increasingly attractive approach, particularly for those lysosomal storage diseases with neurological manifestations. This review summarizes the most recent advances in developing gene therapies for this large and heterogeneous group of disorders.

Lipid-mediated ex vivo gene transfer of viral interleukin 10 in rat lung allotransplantation.

BACKGROUND: Recent studies suggest that viral interleukin 10 suppresses alloimmune response in transplantation and that cationic lipids are one of the most promising nonviral vehicles for gene therapy. The aim of this study was to examine the effect of ex vivo lipid-mediated viral IL10 gene transfer into rat lung allografts on subsequent rejection. METHODS: Male F344 rats (RT1lvl) underwent left lung transplantation with allografts from Brown Norway rats (RT1n). Allografts were transvascularly transfected 15 minutes after harvest with 5 mL of 1:20-diluted (group 1, n = 7) or 1:40-diluted (group 2, n = 6) GL67-pCMVievIL-10 complex. Group 3 (n = 7), serving as the control group, received 1:40-diluted GL67-pCF1-chloramphenicol acetyltransferase complex. All allografts were preserved for 3 hours at 10 degrees C before transplantation. In all groups recipients were killed on postoperative day 5. Transgene expression of viral interleukin 10 was assessed by means of both reverse transcriptase-polymerase chain reaction and immunohistochemistry. Histologic rejection score, allograft gas exchange, exhaled nitric oxide level, and allograft cytokine mRNA expression were also assessed. RESULTS: Dose-dependent transgene expression of viral interleukin 10 was detected by means of both reverse transcriptase-polymerase chain reaction and immunohistochemistry. Allograft gas exchange (PaO2) in groups 1 (114.06 +/- 61.1 mm Hg) and 2 (108.58 +/- 35.7 mm Hg) was significantly better than that in group 3 (66.4 +/- 8.22 mm Hg; P =.020 and P =.023, respectively). The vascular rejection score in group 1 was significantly lower than that in group 3 (P =.032, Kruskal-Wallis test). Exhaled nitric oxide levels in group 2 (5.150 +/- 6.38 ppb) were significantly lower than those in group 3 (13.517 +/- 10.4 ppb; P =.039). Allograft interleukin 2 mRNA expression levels in group 1 (1.123 +/- 0.23 relative units) were significantly lower than those in group 3 (1.753 +/- 0.71 relative units; P =.038 vs group 3). CONCLUSIONS: Lipid-mediated ex vivo viral IL10 gene transfer into rat lung allografts improved graft gas exchange, reduced histologic rejection scores, downregulated graft interleukin 2 mRNA expression, and reduced exhaled nitric oxide levels by postoperative day 5. These results suggest a therapeutic potential of graft viral IL10 gene transfer as an effective immunosuppressive strategy against lung allograft rejection.

High and sustained transgene expression in vivo from plasmid vectors containing a hybrid ubiquitin promoter.

Sustained transgene expression will be required for the successful treatment of most genetic diseases being considered for gene therapy. The initially high levels of expression attained with plasmid DNA (pDNA) vectors containing viral promoters, such as that from cytomegalovirus (CMV), decline precipitously to near-background levels within two to three weeks. Here we constructed pDNA vectors containing the human cellular UBB (encoding ubiquitin B; Ub) promoter and evaluated their expression in the mouse lung. Cationic lipid-pDNA complexes were instilled intranasally (IN) or injected intravenously (IV) into immunodeficient BALB/c mice. Chloramphenicol acetyltransferase (CAT) reporter gene expression from the UBB promoter was initially very low at day 2 post-administration, but by day 35 exceeded the level of expression attained from a CMV promoter vector by four- to ninefold. Appending a portion of the CMV enhancer 5' of the UBB promoter (CMV-Ub) increased CAT expression to nearly that of the CMV promoter and expression persisted in the lung for at least 3 months, with 50% of day 2 levels remaining at day 84. In the liver, expression from the CMV-Ub hybrid promoter was sustained for 42 days. As previous studies have shown that eliminating immunostimulatory CpG motifs in pDNA vectors reduces their toxicity, we constructed a CpG-deficient version of the CMV-Ub vector expressing alpha-galactosidase A, the enzyme deficient in Fabry disease, a lysosomal storage disorder. After IN or IV administration, levels of alpha-galactosidase A from this vector were not only undiminished but increased 500% to 1500% by day 35. Our results indicate that CpG-reduced plasmid vectors containing a CMV-Ub hybrid promoter may provide the long-term expression required for a practical gene therapeutic.

Endobronchial transfection of naked viral interleukin-10 gene in rat lung allotransplantation.

BACKGROUND: Recent studies suggest that viral interleukin-10 (vIL-10) suppresses alloimmune response in transplantation. Tissue mRNA expression of inducible nitric oxide synthase (iNOS) and exhaled nitric oxide (NO) levels have been observed to increase in lung allograft rejection. The aims of this study were to examine the feasibility of vIL-10 gene transfer into rat lung allografts and to investigate its effect on subsequent allograft rejection. METHODS: Male Lewis rats (RT1l) underwent left lung transplantation with allografts from Brown Norway rats (RT1n). The donor rats were endobronchially transfected 2 minutes before harvest with 400 microg (group I, n = 5), 600 microg (group II, n = 5), or 800 microg (group III, n = 5) of naked pCMVievIL-10. Group IV (n = 5) animals, serving as control, received 400 microg of naked pCF1-CAT. All recipients were sacrificed on postoperative day 5. Transgene expression of vIL-10 was assessed by both reverse transcriptase-polymerase chain reaction and immunohistochemistry. Allograft gas exchange, exhaled NO level, histologic rejection score, and mRNA expression of graft cyokines were also assessed. RESULTS: Transgene expression of lung graft vIL-10 was detected by both reverse transcriptase-polymerase chain reaction and immunohistochemistry. The iNOS mRNA expression in groups I, II, and III was significantly lower than that of group IV (p < 0.05, analysis of variance). Exhaled NO levels in groups I, II, and III were significantly lower than in group IV (p < 0.01, analysis of variance). There was no significant difference between groups with respect to gas exchange, peak airway pressure, or histologic rejection score. CONCLUSIONS: It appears that endobronchial transfection of naked vIL-10 plasmid in a rat lung allotransplant model is feasible and suppresses lung iNOS mRNA expression and exhaled NO levels. An association between iNOS upregulation and high exhaled NO levels in lung allograft resection was also noted.

Requirements for effective inhibition of immunostimulatory CpG motifs by neutralizing motifs.

The DNA of bacteria and many viruses contain unmethylated CpG dinucleotides in particular sequence contexts that activate vertebrate immune cells. A subset of these CpG motifs was previously found to oppose the effects of immunostimulatory (CpG-S) motifs and has been termed neutralizing (CpG-N) motifs. Here we show that oligodeoxynucleotides (ODNs) composed of clusters of CpG-N motifs could partially inhibit the induction of interleukin-12 (IK-12) from mouse spleen cells by ODN containing CpG-S motifs. However, non-CpG-containing ODN were also inhibitory, suggesting that neutralization of CpG-S ODNs by CpG-N ODNs in trans was nonspecific. Neutralization of CpG-S motifs by CpG-N motifs in cis was specific, but the degree of inhibition was strongly dependent on the particular CpG-S motif being neutralized, with motifs having an A residue 5' to the CG being much more resistant to inhibition than motifs having a T residue 5' to the CG. The degree of inhibition was dependent on the spacing between the CpG-S and CpG-N motifs, with the ability to neutralize inversely correlating with distance. In addition, whereas ODNs containing extended clusters of CpG-N motifs were nonstimulatory, isolated CpG-N motifs remained stimulatory in most sequence contexts. Finally, CpG-N ODNs were shown to be nonstimulatory when instilled into the lungs of BALB/c mice, but the ability of CpG-N motifs to neutralize CpG-S motifs in cis was not observed. These results show that there are precise and fairly complex interactions between immunostimulatory and inhibitory sequence motifs that govern whether a given DNA is able to activate the vertebrate immune system.

Reduced inflammatory response to plasmid DNA vectors by elimination and inhibition of immunostimulatory CpG motifs.

An inflammatory response is invariably associated with administration of gene transfer complexes composed of cationic lipids and plasmid DNA (pDNA). In the lung, an influx of neutrophils and elevated levels of several proinflammatory cytokines such as TNF-alpha, IFN-gamma, IL-6, and IL-12 characterize this dose-dependent response. The induction of these cytokines was shown previously to be due in part to the presence of unmethylated CpG dinucleotides in the bacterially derived pDNA. We have eliminated 270 of 526 CpG dinucleotides in a reporter plasmid (pCFA-CAT) and tested the inflammatory response to cationic lipid:pDNA complexes containing the modified vector (pGZA-CAT) after intravenous (i.v.) or intranasal (i.n.) delivery into BALB/c mice. Compared to the unmodified vector, the CpG-reduced pGZA-CAT was found to be significantly less immunostimulatory, as the levels of IL-12, IFN-gamma, and IL-6 in the serum 24 h after i.v. delivery were reduced by 40 to 75%. Similar reductions in cytokine levels were also observed in the bronchoalveolar lavage fluids (BALF) after i.n. administration, while the levels of reporter gene expression were not affected by the modifications. We have also investigated known inhibitors of the CpG signaling pathways in order to decrease the inflammatory response. Two such inhibitors, chloroquine and quinacrine, greatly reduced the induction of IL-12 from mouse spleen cells in vitro and inhibited cytokine production in the lung by approximately 50% without affecting gene expression. These results illustrate that use of a less immunostimulatory pDNA vector or inhibitors of CpG immunostimulation can reduce significantly the toxicity associated with cationic lipid:pDNA complexes thereby increasing the therapeutic index of this synthetic gene transfer vector.

Transforming growth factor-beta1 gene transfer ameliorates acute lung allograft rejection.

BACKGROUND: The aim of the current work was to study the feasibility of functional gene transfer using the gene encoding for transforming growth factor-beta1, a known immunosuppressive cytokine, on rat lung allograft function in the setting of acute rejection. METHODS: The rat left lung transplant technique was used in all experiments, with Brown Norway donor rats and Fischer recipient rats. After harvest, left lungs were transfected ex vivo with either sense or antisense transforming growth factor-beta1 constructs complexed to cationic lipids, then implanted into recipients. On postoperative days 2, 5, and 7, animals were put to death, arterial oxygenation measured, and acute rejection graded histologically. RESULTS: On postoperative day 2, there were no differences in acute rejection or lung function between animals treated with transforming growth factor-beta1 and control animals. On postoperative day 5, oxygenation was significantly improved in grafts transfected with the transforming growth factor-beta1 sense construct compared with antisense controls (arterial oxygen tension = 411 +/- 198 vs 103 +/- 85 mm Hg, respectively; P =.002). Acute rejection scores from lung allografts were also significantly improved, corresponding to decreases in both vascular and airway rejection (vascular rejection scores: 2.0 +/- 0. 5 vs 2.8 +/- 0.6; P =.04; airway rejection scores: 1.3 +/- 0.7 vs 2. 3 +/- 0.8, respectively; P =.02). The amelioration of acute rejection was temporary and decreased by postoperative day 7. CONCLUSIONS: The feasibility of using gene transfer techniques to introduce novel functional genes in the setting of lung transplantation is demonstrated. In this model of rat lung allograft rejection, gene transfer of transforming growth factor-beta1 resulted in temporary but significant improvements in lung allograft function and acute rejection pathology.

Increased duration of transgene expression in the lung with plasmid DNA vectors harboring adenovirus E4 open reading frame 3.

For gene therapy to be effective in the treatment of chronic diseases, plasmid DNA (pDNA) vectors that provide persistent expression of therapeutic levels of the transgene product are desirable. Studies in the lung with adenovirus vectors showed that products of the adenovirus E4 region can act both in cis and in trans to increase the duration of expression when transcription of the transgene was under the control of the human cytomegalovirus (CMV) promoter. To determine if these E4-encoded proteins could also effect greater persistence of expression from a nonviral vector, a complex composed of cationic lipid GL-67, a CMV promoter plasmid (pCF1-CAT), and an E4-containing adenovirus vector (Ad2/betagal-4) was instilled into the lungs of BALB/c nu/nu mice. Significant increases in the duration of transgene expression were observed for up to 10 weeks postinstillation compared with expression from mice instilled with control complexes containing an adenovirus vector deleted of most of E4 (Ad2/betagal-2). This effect could also be observed in immunodeficient NIH-rnu rats as well as in immunocompetent BALB/c mice. Studies with CMV promoter mutants indicated that a region proximal to the promoter was necessary for the E4-mediated increase in longevity of expression. In addition to the CMV promoter, a CMV enhancer-human mucin I (MUC-I) hybrid promoter also responded to these E4-encoded proteins with increased persistence of transgene expression, but a human interleukin 8 (IL-8) promoter did not. Ad2/betagal-4 could be replaced by a pDNA vector expressing only the E4 region, indicating that products of the E4 region alone were sufficient in the absence of expression from the rest of the adenovirus genome. Further analysis indicated that the protein encoded by open reading frame 3 (ORF3) alone was sufficient for conferring the increase in persistence of expression. These data indicate that expression of a single protein from the adenovirus genome can significantly improve the duration of transgene expression from pDNA vectors, and increases the feasibility of using nonviral vectors for the treatment of chronic diseases.

Correction of enzymatic and lysosomal storage defects in Fabry mice by adenovirus-mediated gene transfer.

Fabry disease is a recessive, X-linked disorder caused by a deficiency of the lysosomal hydrolase alpha-galactosidase A. Deficiency of this enzyme results in progressive deposition of the glycosphingolipid globotriaosylceramide (GL-3) in the vascular lysosomes, with resultant distension of the organelle. The demonstration of a secretory pathway for lysosomal enzymes and their subsequent recapture by distant cells through the mannose 6-phosphate receptor pathway has provided a rationale for somatic gene therapy of lysosomal storage disorders. Toward this end, recombinant adenoviral vectors encoding human alpha-galactosidase A (Ad2/CEHalpha-Gal, Ad2/CMVHIalpha-Gal) were constructed and injected intravenously into Fabry knockout mice. Administration of Ad2/CEHalpha-Gal to the Fabry mice resulted in an elevation of alpha-galactosidase A activity in all tissues, including the liver, lung, kidney, heart, spleen, and muscle, to levels above those observed in normal animals. However, enzymatic expression declined rapidly such that by 12 weeks, only 10% of the activity observed on day 3 remained. Alpha-galactosidase A detected in the plasma of injected animals was in a form that was internalized by Fabry fibroblasts grown in culture. Such internalization occurred via the mannose 6-phosphate receptors. Importantly, concomitant with the increase in enzyme activity was a significant reduction in GL-3 content in all tissues to near normal levels for up to 6 months posttreatment. However, as expression of alpha-galactosidase A declined, low levels of GL-3 reaccumulated in some of the tissues at 6 months. For protracted treatment, we showed that readministration of recombinant adenovirus vectors could be facilitated by transient immunosuppression using a monoclonal antibody against CD40 ligand (MR1). Together, these data demonstrate that the defects in alpha-galactosidase A activity and lysosomal storage of GL-3 in Fabry mice can be corrected by adenovirus-mediated gene transfer. This suggests that gene replacement therapy represents a viable approach for the treatment of Fabry disease and potentially other lysosomal storage disorders.

Ex vivo transfection of transforming growth factor-beta1 gene to pulmonary artery segments in lung grafts.

OBJECTIVE: Proximal pulmonary artery segment transfection may provide beneficial downstream effects on the whole-lung graft. In this study, transforming growth factor-beta1 was transfected to proximal pulmonary artery segments, and the efficacy of transforming growth factor-beta1 transfection was examined in ischemia-reperfusion injury and acute rejection models of rat lung transplantation. METHODS: In the ischemia-reperfusion injury model, orthotopic left lung transplantation was performed in F344 rats. In group I, the PPAS was isolated and injected with saline solution. In 2 other groups, lipid67:DOPE:sense (group II) or antisense transforming growth factor-beta1pDNA construct (group III) was injected instead of saline solution. After cold preservation at 4 degrees C for 18 hours, lung grafts were implanted. Graft function was assessed 24 hours later. In the acute rejection model, donor lung grafts were harvested. Proximal pulmonary artery segments were injected with saline solution (group I) or sense (group II) or antisense lipid gene construct (group III) and then implanted. Graft function was assessed on postoperative day 5. RESULTS: In the ischemia-reperfusion injury study, there were no significant differences in oxygenation, wet-to-dry weight ratios, graft myeloperoxidase activity, or transforming growth factor-beta1 levels in platelet-poor serum or proximal pulmonary artery segment homogenates. In the acute rejection study, oxygenation was significantly improved in group II receiving transforming growth factor-beta1 (group II vs I and III, 136.0 +/- 32.5 vs 54.0 +/- 9.6 mm Hg and 53.8 +/- 14.8 mm Hg; P =.016 and.016). There were no significant pathologic differences. Transforming growth factor-beta1 concentrations from proximal pulmonary artery segment homogenates in group II were significantly higher compared with controls. CONCLUSIONS: Ex vivo transfection of transforming growth factor-beta1 to proximal pulmonary artery segments did not affect reperfusion injury of lung isografts. In acute rejection, however, ex vivo transfection of transforming growth factor-beta 1 to proximal pulmonary artery segments improved allograft function. This suggests that transfection to proximal pulmonary artery segments exerts beneficial downstream effects on the whole-lung allograft.

Contribution of plasmid DNA to inflammation in the lung after administration of cationic lipid:pDNA complexes.

Cationic lipid-mediated gene transfer to the mouse lung induces a dose-dependent inflammatory response that is characterized by an influx of leukocytes and elevated levels of the cytokines interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma). We have examined the contribution of plasmid DNA (pDNA) to this observed toxicity, specifically the role of unmethylated CpG dinucleotides, which have been previously shown to be immunostimulatory. We report here that complexes of cationic lipid GL-67 and unmethylated pDNA (pCF1-CAT) instilled into the lungs of BALB/c mice induced highly elevated levels of the cytokines TNF-alpha, IFN-gamma, IL-6, and IL-12 in the bronchoalveolar lavage fluids (BALF). In contrast, BALF of animals administered either GL-67 alone or GL-67 complexed with SssI-methylated pDNA contained low levels of these cytokines. Similar results were observed using a plasmid (pCF1-null) that does not express a transgene, demonstrating that expression of chloramphenicol acetyltransferase (CAT) was not responsible for the observed inflammation. The response observed was dose dependent, with animals receiving increasingly higher amounts of unmethylated pDNA exhibiting progressively higher levels of the cytokines. Concomitant with this increase in cytokine levels were also elevated numbers of neutrophils in the BALF, suggesting a possible cause- and-effect relationship between neutrophil influx and generation of cytokines. Consistent with this proposal is the observation that reduction of neutrophils in the lung by administration of antibodies against Mac-1alpha and LFA-1 also diminished cytokine levels. This reduction in cytokine levels in the BALF was accompanied by an increase in transgene expression. In an attempt to abate the inflammatory response, sequences in the pDNA encoding the motif RRCGYY, shown to be most immunostimulatory, were selectively mutagenized. However, instillation of a plasmid in which 14 of the 17 CpG sites were altered into BALF/c mice did not reduce the levels of cytokines in the BALF compared with the unmodified vector. This suggests that other unmethylated motifs, in addition to RRCGYY, may also contribute to the inflammatory response. Together, these findings indicate that unmethylated CpG residues in pDNA are a major contributor to the induction of specific proinflammatory cytokines associated with instillation of cationic lipid:pDNA complexes into the lung. Strategies to abate this response are warranted to improve the efficacy of this nonviral gene delivery vector system for the treatment of chronic diseases.

Basis of pulmonary toxicity associated with cationic lipid-mediated gene transfer to the mammalian lung.

Studies have indicated that although abundant levels of transgene expression could be achieved in the lungs of mice instilled with cationic lipid:pDNA complexes, the efficiency of gene transfer is low. As a consequence, a relatively large amount of the complex will need to be administered to the human lungs to achieve therapeutic efficacy for indications such as cystic fibrosis. Because all cationic lipids exhibit some level of cytotoxicity in vitro, we assessed the safety profile of one such cationic lipid, GL-67, following administration into the lungs of BALB/c mice. Dose-dependent pulmonary inflammation was observed that was characterized by infiltrates of neutrophils, and, to a lesser extent, macrophages and lymphocytes. The lesions in the lung were multifocal in nature and were manifested primarily at the junction of the terminal bronchioles and alveolar ducts. The degree of inflammation abated with time and there were no apparent permanent fibrotic lesions, even in animals that were treated at the highest doses. Analysis of the individual components of the complex revealed that the pulmonary inflammation was primarily cationic lipid-mediated with a minor contribution from the neutral co-lipid DOPE. Associated with the lesions in the lungs were elevated levels of the pro-inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) that peaked at days 1-2 post-instillation but resolved to normal limits by day 14. Total cell counts, primarily of neutrophils, were also significantly elevated in the bronchoalveolar lavage fluids of GL-67:pDNA-treated mice between days 1 and 3 but returned to normal limits by day 14. No specific immune responses were detected against the cationic lipid or plasmid DNA in mice that had been either instilled or immunized with the individual components or complex, nor was there any evidence of complement activation. These studies indicate that a significant improvement in the potency of cationic lipid:pDNA formulations is desirable to minimize the toxicity associated with cationic lipids.

Optimization of plasmid vectors for high-level expression in lung epithelial cells.

Nonviral gene therapy approaches use a plasmid vector to express the desired transgene. We have systematically examined several regulatory elements within plasmid vectors that govern gene expression, e.g., the promoter, enhancer, intron, and polyadenylation signal, by constructing a series of plasmids that differed only in the particular sequence element being evaluated. Of the several promoters and polyadenylation signal sequences that were tested, the human cytomegalovirus (CMV) immediate early gene promoter and the addition of polyadenylation signal sequences from the bovine growth hormone (BGH) gene or rabbit beta-globin gene produced the highest levels of expression in vitro. The inclusion of a hybrid intron 3 to the promoter further increased expression 1.6-fold. The addition of a region of the CMV enhancer 5' to several weak promoters increased expression 8- to 67-fold, and co-transfection with a second plasmid encoding a chimeric transcription factor also enhanced expression. On the basis of these results, the CMV promoter, the hybrid intron, and the BGH polyadenylation signal were selected for consistent high level expression in vitro and in the mouse lung. However, expression was transient, with greater than 60% loss of activity in the first 7 days. This transient expression was not specific to CMV promoter-containing plasmids, because plasmids containing other heterologous promoters showed a similar profile of transient expression in vivo. These comparative analyses begin to provide a basis for the development of optimized expression plasmids for gene therapy of lung diseases.

Detailed analysis of structures and formulations of cationic lipids for efficient gene transfer to the lung.

Cationic lipid-mediated gene transfer of cystic fibrosis transmembrane conductance regulator (CFTR) cDNA represents a promising approach for treatment of cystic fibrosis (CF). Here, we report on the structures of several novel cationic lipids that are effective for gene delivery to the lungs of mice. An amphiphile (#67) consisting of a cholesterol anchor linked to a spermine headgroup in a "T-shape" configuration was shown to be particularly efficacious. An optimized formulation of #67 and plasmid vector encoding chloramphenicol acetyl-transferase (CAT) was capable of generating up to 1 microgram of CAT enzyme/lung following intranasal instillation into BALB/c mice. This represents a 1,000-fold increase in expression above that obtained in animals instilled with naked pDNA alone and is greater than 100-fold more active than cationic lipids used previously for CFTR gene expression. When directly compared with adenovirus-based vectors containing similar transcription units, the number of molecules of gene product expressed using lipid-mediated transfer was equivalent to vector administration at multiplicities of infection ranging from 1 to 20. The level of transgene expression in the lungs of BALB/c mice peaked between days 1 and 4 post-instillation, followed by a rapid decline to approximately 20% of the maximal value by day 7. Undiminished levels of transgene expression in the lung could be obtained following repeated intranasal administration of #67:DOPE:pCF1-CAT in nude mice. Transfection of cells with formulations of #67:DOPE:pCF1-CFTR generated cAMP-stimulated CFTR chloride channel and fluid transport activities, two well-characterized defects associated with CF cells. Taken together, the data demonstrate that cationic lipid-mediated gene delivery and expression of CFTR in CF lungs is a viable and promising approach for treatment of the disease.

Two different mRNAs are transcribed from a single genomic locus encoding the chicken erythrocyte anion transport proteins (band 3).

The chicken erythrocyte anion transport protein (band 3 of the erythrocyte cytoskeleton) is a central component taking part in two widely divergent functions of erythroid cells; it is a primary determinant of cytoskeletal architecture and responsible for electroneutral Cl-/HCO3- exchange across the plasma membrane. To analyze interesting aspects of the developmental regulation of this gene, we have cloned the cDNA and genomic counterparts of the erythroid-specific anion transport protein. We show that a single genetic locus for band 3 encodes two different erythroid cell-specific mRNAs, with different translational initiation sites, which predict polypeptides of sizes very close to those observed in vivo. In vitro translation and immune precipitation of synthetic mRNA derived from one putative fully encoding cDNA clone demonstrate that this clone gives rise to a protein which is identical in size and antigenicity to bona fide chicken erythroid band 3.

Expression of cytoskeletal protein 4.1 during avian erythroid cellular maturation.

We have isolated a cDNA clone encoding part of protein 4.1, an integral component of the erythrocyte cytoskeleton. The recombinant was isolated by immunological screening of a chicken erythroid lambda gt11 cDNA library using a monoclonal antibody directed against protein 4.1. DNA blot analysis shows that the gene is present as a single copy per haploid chicken genome, while RNA blot analysis reveals the presence of a single mRNA of 7 kilobases in reticulocytes. Message of the same size (in reduced amounts) is also present in an erythroleukemic cell line transformed by avian erythroblastosis virus and is also present in vastly reduced quantities in nonerythroid hemopoietic cells. Immunoblotting and immunofluorescence experiments show that a subset of the chicken 4.1 variant proteins is preferentially expressed during in vitro differentiation of chicken erythroleukemic cells. These data indicate that the gene is both actively transcribed and translated during early erythroid cellular maturation.

Isolation of recombinant cDNAs encoding chicken erythroid delta-aminolevulinate synthase.

We report the isolation of cDNA clones encoding delta-aminolevulinate synthase (ALA synthase; EC 2.3.1.37), the first enzyme in the heme biosynthetic pathway in animal cells. The gene was isolated from a chicken erythroid cDNA library prepared in the bacteriophage lambda fusion/expression vector gt11, using rabbit antibody raised against the relatively abundant chicken liver enzyme. The chicken liver and red cell ALA synthase isozymes share substantial crossreactivity to the antibody, thereby allowing isolation of the erythroid-specific gene by using the heterologous antibody in immune screening of the red cell cDNA library. Preliminary analysis documenting the tissue specificity of transcription indicates that the enzyme is encoded by a highly homologous set of messages, which appear to differ in size in various avian tissues. From analysis using strand-specific RNA probes, it appears that the different ALA synthase mRNAs detected may be transcribed from a family of genes that are closely related in nucleotide sequence and are each regulated in a developmentally specific manner.