Lentivirus-mediated gene transfer to the rat, ovine and human cornea.

Gene therapy of the cornea shows promise for modulating corneal transplant rejection but the most appropriate vector for gene transfer has yet to be determined. We investigated a lentiviral vector (LV) for its ability to transduce corneal endothelium. A lentivector expressing enhanced yellow fluorescent protein (eYFP) under the control of the Simian virus type 40 early promoter (LV-SV40-eYFP) transduced 80-90% of rat, ovine and human corneal endothelial cells as detected by fluorescence microscopy. The kinetics of gene expression varied among species, with ovine corneal endothelium showing a relative delay in detectable reporter gene expression compared with the rat or human corneal endothelium. Vectors containing the myeloproliferative sarcoma virus promoter or the phosphoglycerate kinase promoter were not significantly more effective than LV-SV40-eYFP. The stability of eYFP expression in rat and ovine corneas following ex vivo transduction of the donor cornea was assessed following orthotopic corneal transplantation. Following transduction ex vivo, eYFP expression was maintained in corneal endothelial cells for at least 28 days after corneal transplantation in the sheep and >60 days in the rat. Thus, rat, ovine and human corneal endothelial cells were efficiently transduced by the LV, and gene expression appeared stable over weeks in vivo.

Codon-optimized reading frames facilitate high-level expression of the HIV-1 minor proteins.

We have constructed reading frames for the HIV-1 YU-2 minor proteins Vpr, Vpu, Vif and Nef that are codon-optimized for high-level expression in mammalian cells. We show that, in the absence of the Rev/Rev-response element system, these codon-optimized reading frames result in greatly increased levels of expression of the corresponding proteins in cell culture systems when compared with the native reading frame. Northern blot analysis shows that the increase in expression found with the codon-optimized reading frames is largely owing to increased steady-state mRNA levels.

Optimisation of a multipartite human immunodeficiency virus based vector system; control of virus infectivity and large-scale production.

BACKGROUND: We have previously described a five-plasmid HIV-1 vector system that utilises a codon-optimised gagpol gene. While this system was shown to be safer than systems using proviral type helpers, the titre of virus produced was relatively low. Therefore, a process of optimising all aspects of virus production was initiated. METHODS: A systematic approach was taken to the optimisation of virus production by transient expression using a five-plasmid packaging system. Codon-manipulation was used to reduce homology between helper and vector constructs. Ultrafiltration and ultracentrifugation were used for large-scale virus production. RESULTS: We describe codon-optimised reading frames for Tat and Rev and the optimisation of virus production. The optimisation process resulted in an increase in virus titre of 7- to 8-fold. Several other approaches to increasing viral titre described by others proved ineffective in our system after it had been optimised. In addition, we show that by varying the ratio of the GagPol helper construct to vector, the infectivity of the virus could be controlled. The use of a novel codon-optimised HIV-1 GagPol expression construct with reduced homology to vector sequences significantly reduced transfer of gagpol sequences to transduced cells. Virus could be collected in serum-free medium without a significant loss of titre, which facilitated subsequent processing. Processing using a combination of ultrafiltration and ultracentrifugation allowed efficient and rapid processing of litre volumes of virus supernatant. CONCLUSIONS: By taking a systematic approach to optimising all aspects of our five-plasmid lentiviral vector system we improved titre, safety, large-scale production, and demonstrated that infectivity could be specifically controlled.

Can the use of HIV-1 derived gene transfer vectors for clinical application be justified?

Vectors derived from human immunodeficiency virus type 1 (HIV-1) are an attractive option for many gene therapy applications as they can transduce non-cycling cell populations, and can integrate their genome into the host cell chromosome. The rationale underlying the design of most retroviral vector systems is to segregate the viral cis sequences, which are required for transfer of the viral genome, from the trans sequences that encode viral proteins. This allows the efficient production of replication incompetent virus and has been successfully applied to the generation of HIV-1 vectors. Nonetheless, the possibility that recombination events in the vector production system can generate replication-competent virus, combined with the pathogenic nature of HIV-1, raises major bio-safety issues. Numerous HIV-1 vectors have now been reported, with each generation significantly improved in ways designed to reduce the risk of replication-competent virus being produced. However, progress in vector design needs to be complemented by the development of methods for the quantitation of the probability of replication competent virus being produced. Assaying individual events in the multi-step pathway that can lead to the production of replication-competent virus, rather than relying on the detection of replication-competent virus per se, will be important for quality control purposes. This review will specifically examine the approaches to HIV-1 vector design that have been postulated as increasing bio-safety, possible methods for evaluating bio-safety and whether these approaches are likely to be sufficient to overcome resistance to the use of HIV-1 for clinical application. In addition, we discuss the possible justifications for developing vectors from lentiviruses other than HIV-1.

An improved beta-galactosidase reporter gene.

The coding sequence for the E. coli beta-galactosidase gene was codon-optimised for expression in mammalian cells. When expressed in mammalian cells the codon-optimised gene results in the expression of beta-galactosidase at levels 15-fold higher than those resulting from an analogous construct containing the native E. coli gene sequence. RNA analysis suggests the enhancement of beta-galactosidase expression is due both to enhanced transcript stability and increased translational efficiency. When used in a lentiviral construct the codon-optimised gene results in an approximately five-fold increase in apparent titre, as determined by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining, in comparison to an analogous construct containing the native E. coli gene. Southern blot analysis shows this is due to an increased efficiency of detection of transduced cells. In addition, codon-optimisation results in the elimination of several cryptic splice acceptor sites that are present in the native E. coli gene sequence. In a lentiviral vector containing a 5' splice donor the use of the codon-optimised gene in place of the native E. coli beta-galactosidase gene resulted in increased amounts of un-spliced, full-length genomic RNA. Therefore, as a marker/reporter gene in mammalian cells the codon-optimised beta-galactosidase gene has a number of advantages over the native E. coli gene sequence. A variant of the codon-optimised beta-galactosidase gene sequence that includes an effective nuclear localisation signal was also made.

Rational development of a HIV-1 gene therapy vector.

BACKGROUND: HIV-1 provides an attractive option as the basis for gene transfer vectors due to its ability to stably transduce non-cycling cell populations. In order to fully utilise the promise of HIV-1 as a vector it is important that the effects of viral cis sequence elements on vector function are carefully delineated. METHODS: In this study we have systematically evaluated the effect of various cis elements from the HIV-1 YU-2 genome that have been implicated as either affecting vector performance, or HIV-1 replication, on the efficiency of vector production (titre and infectivity). As a measure of the relative safety of vectors their propensity to inadvertently transfer the gagpol gene to transduced cells was assessed. RESULTS: Sequences that were found to increase vector titre were from the 5' end of the gag gene, from the 5' and 3' ends of the env gene, from immediately upstream of the polypurine tract, and the central polypurine tract. The substitution of the HIV-1 RRE with heterologous RNA transport elements, or the deletion of the RRE, resulted in greatly reduced vector titres. RNA analysis suggested that the role of the Rev/RRE system extends beyond simply acting as an RNA nuclear export signal. The relative safety of different vector designs was compared and an optimal construct selected. CONCLUSIONS: Based on our results we have constructed a vector that is both more efficient, and has better safety characteristics, than the widely used pHR' HIV-1 vector construct.

Helper plasmids for production of HIV-1-derived vectors.

Vectors derived from human immunodeficiency virus type 1 (HIV-1) appear an attractive option for many gene therapy applications. This is due to their ability to transduce noncycling cell populations and to integrate their genome into the host cell chromosome, resulting in the stable genetic modification of the transduced cell. These properties have permitted the direct in vivo transduction of several tissues, including the central nervous system, retina, and liver. However, the pathogenic nature of HIV-1 has raised considerable concerns about the safety of such vector systems. To help address these concerns, we have expressed each of the primary transcriptional units encoding trans functions relevant for vector production in individual plasmid constructs. The gag-pol gene sequence was codon-optimized for expression in mammalian cells resulting in high level Rev/Rev-response element (RRE)-independent expression. Codon optimization of gag-pol also reduces sequence homology with vectors containing gag gene sequences, which results in reduced transfer of biologically active gag-pol sequences to transduced cells. Furthermore, the vif reading frame overlapping the 3' end of the pol coding sequence is destroyed by codon optimization. We have also shown that the Gag and Gag-Pol polyproteins can be efficiently expressed from separate transcriptional units. This has enabled the removal of a cis-acting viral element, the gag-pol translational frameshift sequence, from the vector/packaging system and prevents detectable transfer of biologically active sequences equivalent to the gag-pol gene to transduced cells.

Recombinant canine alpha-l-fucosidase: expression, purification, and characterization.

Canine fucosidosis has proven to be an excellent large animal model both for the equivalent human disorder and, in more general terms, for the central nervous system pathology found in many of the lysosomal storage disorders. Most importantly studies in this animal model were among the first to convincingly show that bone marrow transplantation could successfully modify the course of clinical central nervous system disease and to define some of the important parameters for successful treatment. In order to evaluate other, more generally applicable routes to treatment of central nervous system disease in the lysosomal storage disorders we have expressed recombinant canine alpha-l-fucosidase (rcFUC) in Chinese hamster ovary and Madin-Darby canine kidney cells to levels of between 2 and 13 mg/liter of culture medium and purified the enzyme to apparent homogeneity by affinity chromatography on fucosylamine-linked agarose. rcFUC is composed of subunits of M(r) 50 kDa and the native enzyme is a homotrimer of M(r) 156 kDa. Kinetic properties of rcFUC were similar to those of FUC isolated from both human and dog liver. rcFUC was shown to be effective in correcting the storage phenotype of human fucosidosis cells after endocytosis via the mannose-6-phosphate-receptor-mediated pathway. It was also shown to degrade fucosylated storage products isolated from affected dog brain. The availability of large amounts of rcFUC will allow us to explore ways of extending the proven efficacy of enzyme replacement therapy to the treatment of central nervous system pathology using the fucosidosis dog as a model system.

Sulphamidase.

Sulphamidase is one of four lysosomal proteins whose deficiency clinically manifests as Sanfilippo syndrome. Deficiency of sulphamidase results in the lysosomal storage of the glycosaminoglycan (GAG) heparan sulphate (HS) and is termed mucopolysaccharidosis type IIIA (MPS IIIA). Sulphamidase catalyses the hydrolysis of an N-linked sulphate from the nonreducing terminal glucosaminide residue of HS (Fig. 1). It is unique among the known lysosomal sulphatases involved in GAG degradation in that it is an N-sulphatase, all the others being O-sulphatases. Purification of sulphamidase from human liver has facilitated the amino-terminal sequencing of the protein and hence the isolation of cDNA and genomic clones for sulphamidase. This has in turn made possible a range of further studies aimed at better diagnosis, treatment and understanding of MPS IIIA.

Regulation of N-acetylgalactosamine 4-sulfatase expression in retrovirus-transduced feline mucopolysaccharidosis type VI muscle cells.

As a preliminary step toward muscle-mediated gene therapy in the mucopolysaccharidosis (MPS) type VI cat, we have analyzed the transcriptional regulation of feline N-acetylgalactosamine 4-sulfatase (f4S) gene expression from various retroviral constructs in primary cultures of muscle cells. Two retroviral constructs were made containing the f4S cDNA under the transcriptional control of the human polypeptide chain-elongation factor 1alpha (EF1alpha) gene promoter or the cytomegalovirus (CMV) immediate-early promoter. Two further retroviral constructs were made with the murine muscle creatine kinase (mck) enhancer sequence upstream of the internal promoter. Virus made from each construct was used to transduce feline MPS VI myoblasts. The mck enhancer significantly upregulated f4S gene expression from both the EF1alpha promoter and the CMV promoter in transduced myoblasts and in differentiated myofibers. The highest level of 4S activity was observed in myoblasts and myofibers transduced with the retroviral construct Lmckcmv4S, in which the f4S gene is under the transcriptional regulation of the mck enhancer and CMV immediate-early promoter. Lmckcmv4S-transduced myofibers demonstrated correction of glycosaminoglycan storage and contained a 58-fold elevated level of 4S activity compared with normal myofibers. Recombinant f4S secreted from Lmckcmv4S-transduced myofibers was endocytosed by feline MPS VI myofibers, leading to correction of the biochemical storage phenotype.

Evaluation of fibroblast-mediated gene therapy in a feline model of mucopolysaccharidosis type VI.

Fibroblast-mediated ex vivo gene therapy was evaluated in the N-acetylgalactosamine 4-sulfatase (4S) deficient mucopolysaccharidosis type VI (MPS VI) cat. Skin biopsies were obtained at birth from severely affected MPS VI kittens and used to initiate fibroblast outgrowths for retroviral transduction with the 4S cDNA. 4S gene expression in transduced cells was under the transcriptional control of the MoMLV long terminal repeat promoter or the cytomegalovirus (CMV) immediate-early promoter. Characterisation of gene-transduced fibroblasts demonstrated the cells to be over-expressing 4S activity. Twenty-four to forty million autologous, gene-corrected fibroblasts were implanted under the renal capsule of three MPS VI kittens at 8-16 weeks of age. Transient, low levels of 4S activity were detected in peripheral blood leukocytes shortly after implantation but were not detectable within 3-8 weeks' post-implantation. Long-term biochemical and clinical evaluation of these cats demonstrated identical disease progression to that previously described in untreated, clinically severe MPS VI cats.

Mild feline mucopolysaccharidosis type VI. Identification of an N-acetylgalactosamine-4-sulfatase mutation causing instability and increased specific activity.

The missense mutation, L476P, in the N-acetylgalactosamine 4-sulfatase (4S) gene, has previously been shown to be associated with a severe feline mucopolysaccharidosis type VI (MPS VI) phenotype. The present study describes a second mutation, D520N, in the same MPS VI cat colony, which is inherited independently of L476P and is associated with a clinically mild MPS VI phenotype in D520N/L476P compound heterozygous cats. Biochemical and clinical assessment of L476P homozygous, D520N/L476P compound heterozygous, and D520N homozygous cats demonstrated that the entire range of clinical phenotypes, from severe MPS VI, to mild MPS VI, to normal are clustered within a narrow range of residual 4S activity from 0. 5% to 4.6% of normal levels. When overexpressed in CHO-KI cells, the secreted form of D520N 4S was inactivated in neutral pH conditions. In addition, intracellular D520N 4S protein was rapidly degraded and corresponded to 37%, 14.5%, and 0.67% of normal 4S protein levels in the microsomal, endosomal, and lysosomal compartments, respectively. However, the specific activity of lysosomal D520N 4S was elevated 22. 5-fold when compared with wild-type 4S. These results suggest that the D520N mutation causes a rapid degradation of 4S protein. The effect of this is partially ameliorated as a result of a significant elevation in the specific activity of mutant D520N 4S reaching the lysosomal compartment.

Receptor mediated binding of two glycosylation forms of N-acetylgalactosamine-4-sulphatase.

The lysosomal storage disorders are a group of inherited metabolic diseases each characterised by a relative or absolute deficiency of one or more of the lysosomal proteins involved in the hydrolysis of glycoconjugates or in the transport of the resulting product. Enzyme replacement therapies are under consideration for a number of these disorders and are based on the in vitro observation that cells from affected patients can be corrected by addition of exogenous enzyme. In this study, two glycosylation variants of the lysosomal enzyme N-acetylgalactosamine-4-sulphatase (4S) (the deficiency of which causes Mucopolysaccharidosis (MPS) type VI, (Maroteaux-Lamy syndrome) were made by expression of 4S cDNA in both wild type chinese hamster ovary (CHO-K1), and Lec1 (N-acetylglucosaminyltransferase I deficient CHO-K1) cells. Differences in the glycosylation pattern of the two enzyme forms were demonstrated with endoglycosidase H and N-glycosidase F digestions. The receptor mediated binding of these two forms of 4S to two cell types, human skin fibroblasts and rat alveolar macrophages, was then analysed. We have shown that both enzyme forms bind to the mannose-6-phosphate receptor on human skin fibroblasts with equal affinity demonstrating that the degree of phosphorylation of mannose residues in the two forms is similar. However, using rat alveolar macrophages, we found that the binding/uptake of the two enzymes differs considerably. These results show that differences in glycosylation of lysosomal enzymes can be an important factor in altering enzyme uptake by different cell types. Thus, producing carbohydrate modification variants in this way may be useful for altering the distribution of exogenous enzyme in vivo.

Determination of cell origin after marrow transplantation in canines by polymerase chain reaction and quantitation of the ZFY/ZFX genes.

BACKGROUND: In order to follow the course of bone marrow engraftment in dogs, and to determine the presence, and percentage, of donor-derived cells in other canine tissues, a simple and fast method of determining cell origin after sex-mismatched bone marrow transplantation was developed. METHODS: Using universal primers, fragments from genomic DNA corresponding to ZFX and ZFY genes were amplified by polymerase chain reaction. A restriction fragment length polymorphism, combined with densitometric analysis, was then used to distinguish and quantitate ZFY and ZFX sequences. Unknown samples were analyzed against standards of known mixtures of male and female DNA. RESULTS: Canine ZFY and ZFX genes were clearly resolved after amplification, digestion with HaeIII, and denaturing polyacrylamide gel electrophoresis. Microchimerism could be detected in male and female dog DNA samples derived from a range of fresh and frozen tissues including spleen, testicle, and the central nervous system. The levels of chimerism determined using this method were in either agreement with results obtained by karyotyping or more sensitive, with a detection limit of 0.4% compared with 1-2%. CONCLUSIONS: Polymerase chain reaction/restriction fragment length polymorphism detection of the ZFY and ZFX genes was found to be simple, accurate, and reliable for assessing engraftment in dogs. When compared with cytogenetic analysis, this method was found to be faster to perform, more capable of detecting lower levels of microchimerism, and useful for detecting donor-derived cells in stored specimens and in tissues other than peripheral blood or bone marrow.

Recombinant human sulphamidase: expression, amplification, purification and characterization.

Mucopolysaccharidosis type IIIA (MPS IIIA, Sanfilippo A syndrome) is a lysosomal storage disease that causes a profound neurological deterioration. The disorder is caused by a deficiency of the lysosomal enzyme sulphamidase which is a requisite for the degradation of heparan sulphate. To facilitate the development of enzyme-replacement strategies for MPS IIIA patients, we have constructed a high-level expression system for recombinant human sulphamidase in Chinese hamster ovary (CHO) cells. An expression construct containing a methotrexate-resistant dihydrofolate reductase (DHFR) gene allowed amplification of expression levels from less than 1 mg of sulphamidase per litre of culture medium to approx. 15 mg/l. Unlike many cell lines made by gene amplification in DHFR-deficient CHO cells, and utilizing the normal DHFR gene, these cell lines appeared to be stable in the absence of selective pressure. Recombinant human sulphamidase was purified from unamplified and amplified cell lines. The native enzyme was found to be a dimer of 115 kDa. Denaturing and reducing SDS/PAGE revealed a subunit size of 62 kDa. Kinetic analysis demonstrated that the recombinant enzyme had broadly similar kinetic characteristics to sulphamidase purified from liver. Recombinant human sulphamidase was able to correct the storage phenotype of MPS IIIA fibroblasts after endocytosis via the mannose-6-phosphate receptor.

Feline mucopolysaccharidosis type VI: correction of glycosaminoglycan storage in myoblasts by retrovirus-mediated transfer of the feline N-acetylgalactosamine 4-sulfatase gene.

Mucopolysaccharidosis type VI (MPS VI) is an autosomal recessive lysosomal storage disorder characterised by the deficiency of N-acetylgalactosamine 4-sulfatase (4S). MPS VI has also been described in the cat. As an initial step toward muscle-mediated gene therapy in the MPS VI cat, we have made two retroviral constructs (pLf4S and pLf4SSN) that transduce the feline 4S gene. Both constructs were designed to express the feline 4S sequence from the viral long terminal repeat promoter. In addition pLf4SSN expressed the neomycin resistance gene from the SV40 early promoter. Amphotrophic virus was produced for each construct and used to transduce feline MPS VI myoblasts. Lf4S- and Lf4SSN-transduced MPS VI feline myoblasts demonstrated correction of glycosaminoglycan storage and contained 55-fold and 3.5-fold elevated levels of 4S activity when compared with normal feline myoblasts respectively. Recombinant feline 4S (rf4S) secreted by Lf4S-transduced MPS VI myoblasts was shown to be endocytosed by MPS VI feline cells via the mannose-6-phosphate receptor system, leading to metabolic correction. The results from this study demonstrate that muscle-mediated gene replacement therapy may be a viable method for achieving circulating levels of recombinant f4S (rf4S) in the MPS VI cat.

Expression, purification and characterization of recombinant caprine N-acetylglucosamine-6-sulphatase.

Mucopolysaccharidosis type IIID or Sanfilippo D syndrome is a lysosomal storage disorder caused by the deficiency of N-acetylglucosamine-6-sulphatase (Glc6S). In addition to human patients, a Nubian goat with this disorder has been described and the caprine Glc6S (cGlc6S) cDNA cloned. In this study, the full-length cGlc6S cDNA was inserted into the expression vector, pEFNeo, which placed the cGlc6S cDNA under the transcriptional control of the human polypeptide chain elongation factor promoter. The pEFNeo expression vector also contains the human growth hormone polyadenylation signal and the genes encoding resistance to ampicillin and G418. The cGlc6S expression construct was electroporated into Chinese hamster ovary (CHO-K1) cells, and stably transfected clones were isolated. One clone, CHOrcGlc6S.17, which secreted the highest Glc6S activity into the culture medium, was selected and cultured in cell factories. The secreted recombinant cGlc6S (rcGlc6S) precursor was purified to homogeneity from conditioned medium by a two-column procedure which consisted of a Cu2+-chelating Sepharose column followed by TSK G3000SW gel filtration. The native molecular mass of rcFlc6S was estimated to be 102 kDa and the subunit size was 94 kDa. The kinetic properties of cGlc6S were similar to those of human Glc6S isolated from liver. rcGlc6S was endocytosed by fibroblasts from patients with mucopolysaccharidosis type IIID via the mannose 6-phosphate receptor-mediated pathway resulting in correction of the storage phenotype of these cells.

Immortalization and characterization of a cell line exhibiting a severe multiple sulphatase deficiency phenotype.

Multiple sulphatase deficiency (MSD) is a rare genetic defect that causes a simultaneous deficiency of all known sulphatases. All available evidence suggests that the deficient gene product is normally responsible for the post-translational modification of a conserved cysteine residue to 2-amino-3-oxopropionic acid and that this modification is necessary for sulphatase activity. MSD often has an enzymically mild phenotype, with significant levels of residual sulphatase activity being detectable. Here we identify an MSD cell line in which the residual activity of the sulphatases assayed was generally very low. To characterize the phenotype of this cell line further, immortalized lines were established after transformation with simian virus 40 (SV40) T antigen. Immortalized cell lines representing normal and MSD phenotypes were then transduced with a retroviral vector carrying the gene encoding human N-acetylgalactosamine-4-sulphatase. Analysis of N-acetylgalactosamine-4-sulphatase protein synthesis and enzyme activity showed that transduced cell lines expressed large amounts of enzyme and that the specific activity of this enzyme was approx. 0.5-1.5% of normal, confirming that this cell line defines a severe phenotype for MSD. N-Acetylgalactosamine-4-sulphatase purified from a transduced MSD cell line seemed normal on denaturing PAGE. Kinetic analysis of the purified enzyme suggests that the residual activity is due to small amounts of normal enzyme rather than unmodified enzyme with low levels of residual activity. These cell lines and the availability of large amounts of inactive N-acetylgalactosamine-4-sulphatase from MSD cells should facilitate the further study of this disorder.

Canine fucosidosis: a model for retroviral gene transfer into haematopoietic stem cells.

Severe progressive fatal neurological degeneration occurs in fucosidosis, a storage disease. Bone marrow transplantation into affected dogs has shown that haematopoietic stem cells can provide enzyme producing daughter cells to the central nervous system, altering disease course. This makes canine fucosidosis an ideal large animal model for gene therapy. Fucosidosis affected allogeneic or autologous canine marrow was transduced ex vivo by cocultivation, then transplanted into fucosidosis affected dogs conditioned with total lymphoid irradiation. The vectors were Moloney murine leukaemia virus based. Transduction efficiency was increased with multiple cytokines in short term marrow culture. Despite high levels of transduction, proviral sequence was detected 2 months post transplant in only one dog. Early or total graft failure occurred in all transplants. We believe lack of engraftment could be caused by differentiation or change of repopulating ability of marrow cells occurring with multiple cytokine mixes in culture media.