Mucopolysaccharidosis (MPS) VI is due to a deficiency in the activity of N-acetylgalactosamine 4-sulfatase (4S), also known as arylsulfatase B. Previously, retroviral vector (RV)-mediated neonatal gene therapy reduced the clinical manifestations of MPS I and MPS VII in mice and dogs. However, sulfatases require post-translational modification by sulfatase-modifying factors. MPS VI cats were injected intravenously (i.v.) with a gamma RV-expressing feline 4S, resulting in 5 +/- 3 copies of RV per 100 cells in liver. Liver and serum 4S activity were 1,450 +/- 1,720 U/mg (26-fold normal) and 107 +/- 60 U/ml (13-fold normal), respectively, and were directly proportional to the liver 4S protein levels for individual cats. This study suggests that sulfatase-modifying factor (SUMF) activity in liver was sufficient to result in active enzyme despite overexpression of 4S. RV-treated MPS VI cats achieved higher body weights and longer appendicular skeleton lengths, had reduced articular cartilage erosion, and reduced aortic valve thickening and aortic dilatation compared with untreated MPS VI cats, although cervical vertebral bone lengths were not improved. This demonstrates that therapeutic expression of a functional sulfatase protein can be achieved with neonatal gene therapy using a gamma RV, but some aspects of bone disease remain difficult to treat.

Although gene therapy has reduced manifestations of genetic diseases, immune responses can abrogate the effect. One approach to inducing tolerance is to perform gene transfer in newborns when the immune system is immature. We demonstrate here that the dose of retroviral vector (RV) is important in mice, as mucopolysaccharidosis I (MPS I) mice that received neonatal intravenous gene therapy with a high dose of a canine alpha-L-iduronidase (cIDUA)-expressing RV had stable expression, while those that received a low dose did not. It was unclear, however, if neonatal transfer with any dose could induce tolerance in large animals. Therefore, newborn MPS I cats were injected intravenously with the RV expressing cIDUA. Although this resulted in high serum IDUA activity due to secretion by transduced cells, expression fell due to a CTL response. Cats that transiently received the immunosuppressive agent CTLA4-Ig did not develop a CTL response. In contrast, MPS I dogs, which can respond immunologically to canine IDUA, had stable serum IDUA activity after neonatal gene therapy. We conclude that cats, but not dogs, mount a potent CTL response to canine IDUA after neonatal gene therapy, which can be prevented with transient CTLA4-Ig.

Although gene therapy has reduced manifestations of genetic diseases, immune responses can abrogate the effect. One approach to induce tolerance is to perform gene transfer in newborns when the immune system is immature. This has allowed a therapeutic response to be achieved for lysosomal storage diseases and hemophilia in mice. However, it is unclear if this approach would be effective in large animals with more-mature immune systems at birth, and it is unlikely that this will be effective in humans. Immune responses will likely pose a tremendous hurdle for patients with null mutations, and identification of a large animal model with a sufficiently mature immune system at birth should facilitate preclinical studies to identify ways to block an expected immune response in humans. Gene therapy experiments were performed in mucopolysaccharidosis I (MPS I) cats, which are deficient in the lysosomal enzyme α-L-iduronidase (IDUA) due to a 3 bp deletion. An amphotropic retroviral vector (RV) expressing the canine IDUA was injected IV into newborn cats at 10(9) or 10(10) TU/kg. The canine protein differs from the feline protein at 130 positions of 653 aa. Expression can be followed by analysis of enzyme activity in serum, as some of the enzyme produced by cells is secreted into blood. Cats achieved high serum activity that averaged 286+/−416 (SD; N=7) U/ml and 2355+/−2763 (SD) U/ml at 1 month after gene transfer for the low and high RV dose, respectively. However, all animals lost expression by 3 months, which was associated with the development of cytotoxic T lymphocytes (CTLs) that could kill autologous cIDUA-expressing fibroblasts, and with a marked decrease in RV RNA sequences in the liver. In contrast, antibodies to cIDUA did not develop. In an attempt to block a CTL response, two cats were treated with the immunosuppressive agent CTLA4- Ig for 2 weeks around the time of transduction with the high dose of RV. CTLA4-Ig binds to CD80 or CD86, and prevents them from stimulating an immune response by activating CD28 on the surface of lymphocytes. These cats achieved 187+/−161 U/ml of IDUA activity in serum, which has been stable for 10 months to date, and did not develop a CTL response These data demonstrate that CTLA4-Ig can prevent a CTL response after neonatal gene therapy. Factors that reduce the efficiency of the immune system in newborn mice and/or humans include reduced secretion of cytokines by lymphocytes, and reduced levels of the co-stimulatory partners CD40 ligand and CD40. In contrast, lymphocytes from newborn humans have normal levels of CD80/CD86 and CD28. We hypothesize that the CD80/CD86 interaction with CD28 may be of particular importance in the newborn period in cats, explaining why transient CTLA4-Ig is so effective at inducing tolerance. The cat appears to be an excellent pre-clinical model for neonatal gene therapy.

Mucopolysaccharidosis I (MPS I) due to deficient alpha-L-iduronidase (IDUA) activity results in accumulation of glycosaminoglycans in many cells. Gene therapy could program liver to secrete enzyme with mannose 6-phosphate (M6P), and enzyme in blood could be taken up by other cells via the M6P receptor. Newborn MPS I mice were injected with 10(9) (high dose) or 10(8) (low dose) transducing units/kg of a retroviral vector (RV) expressing canine IDUA. Most animals achieved stable expression of IDUA in serum at 1240 +/- 147 and 110 +/- 31 units/ml, respectively. At 8 months, untreated MPS I mice had aortic insufficiency, increased bone mineral density (BMD), and reduced responses to sound and light. In contrast, MPS I mice that received high-dose RV had normal echocardiograms, BMD, auditory-evoked brain-stem responses, and electroretinograms. This is the first report of complete correction of these clinical manifestations in any model of mucopolysaccharidosis. Biochemical and pathologic evaluation confirmed that storage was reduced in these organs. Mice that received low-dose RV and achieved 30 units/ml of serum IDUA activity had no or only partial improvement. We conclude that high-dose neonatal gene therapy with an RV reduces some major clinical manifestations of MPS I in mice, but low dose is less effective.

Background/Aims:Hepatocyte replication can be induced in vivo by hepatocyte growth factor (HGF), which might be used for gene therapy or to promote liver regeneration. However, the biochemical steps critical for this process are not clear. C/EBP beta and C/EBP alpha are liver-enriched transcription factors that induce and inhibit hepatocyte replication, respectively. Because of their role in hepatocyte replication, this study examined the effect of HGF upon C/EBP proteins in vivo.Methods: Rats were treated with HGF, and the effect upon C/EBPs was evaluated in liver extracts. Normal or C/EBP beta-deficient mice were treated with HGF, and the effect upon hepatocyte replication was determined.Results: HGF had no effect in rat liver upon C/EBP alpha or C/EBP beta mRNA, nuclear protein, or nuclear DNA binding activity. However, HGF increased phosphorylated p90-RSK and ERK to18- and 3-fold normal, respectively. These kinases phosphorylate C/EBP beta and increase its transcriptional activity. The percentage of hepatocytes that replicated in C/EBP beta-deficient mice after HGF administration was only 1.1%, which was lower than the value of 6.6% for hepatocytes from HGF-treated normal mice (P=0.005).Conclusions: C/EBPP contributes to the induction of hepatocyte replication in response to HGF in rodents, which is likely due to post-translational modifications. (c) 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease due to deficient activity of beta-glucuronidase (GUSB) that results in accumulation of glycosaminoglycans in many organs. We have previously reported that neonatal intravenous injection of a gamma retroviral vector (RV) expressing canine GUSB resulted in transduction of hepatocytes, high levels of GUSB modified with mannose 6-phosphate in blood, and reduction in disease manifestations in the heart, bone, and eye. However, it was unclear if liver was the only site of expression, and the effect upon other organs was not assessed. We demonstrate here that blood cells from these RV-treated MPS VII dogs had substantial copies of RV DNA, and expressed the RNA at 2% of the level found in liver. Therefore, expression of GUSB in blood cells may synergize with uptake of GUSB from blood to reduce storage in organs. The RV-treated dogs had marked biochemical and pathological evidence of reduction in storage in liver, thymus, spleen, small intestines, and lung, and partial reduction of storage in kidney tubules. The brain had 6% of normal GUSB activity, and biochemical and pathological evidence of reduction in storage in neurons and other cell types. Thus, this neonatal gene therapy approach is effective and might be used in humans if it proves to be safe. Both secretion of enzyme into blood by hepatocytes, and expression in blood cells that migrate into organs, may contribute to correction of disease.

Top of pageAbstract Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease (LSD) due to deficient |[aacute]|-L-iduronidase (IDUA) activity. Although neonatal gene therapy with a retroviral vector (RV) expressing canine IDUA (cIDUA) resulted in stable expression in most mice and marked improvement in clinical manifestations, most patients with MPS I are not diagnosed at birth. It will therefore be necessary to determine if gene therapy can be effective in older animals. Adult MPS I mice were transiently treated with hepatocyte growth factor at 6 weeks of age to induce hepatocyte replication, which was followed by injection of hAAT-cIDUA-WPRE during the period of hepatocyte replication. hAAT-cIDUA-WPRE is an amphotropic gamma RV with the liver-specific human |[aacute]|1-antitrypsin promoter upstream of the canine IDUA cDNA, although expression can also derive from the long terminal repeat of the RV in non-hepatic cells. Although most of the IDUA made by hepatocytes is transported to the lysosome after modification with mannose 6-phosphate, some enzyme is secreted into blood where it can be taken up by other organs. Eight mice achieved serum IDUA activity of 97 |[plusmn]|19 U/ml at 1 week after transduction. However, activity fell to <1 U/ml at 3 weeks or later, which was associated with a cytotoxic T lymphocyte response. No anti-cIDUA antibodies were produced. A potent CTL response without an antibody response is different from our previous results with RVs expressing secreted coagulation proteins in adults, suggesting that lysosomal enzymes may be more likely to induce a CTL response than secreted proteins. In an attempt to block this CTL response to cIDUA, two mice were treated with 4 doses of human CTLA4-Ig of 25 mg/kg over 2 weeks starting just prior to gene transfer. CTLA4-Ig is an immunosuppressive agent that binds to CD80 and CD86 and prevents them from activating CD28, an important co-stimulatory molecule on the surface of lymphocytes. These mice achieved 104 |[plusmn]|24 U/ml of serum IDUA that was maintained longer, but fell to <10 U/ml at 3 months or later. This suggests that transient CTLA4-Ig can delay, but not prevent, a CTL response to cIDUA. The final experiment gave CTLA4-Ig for a prolonged period. Five mice received 25 mg/kg twice a week for a month, once a week for a month, and are currently receiving it once a month. All mice have maintained stable expression of cIDUA in serum at 94 |[plusmn]|10 U/ml for up to 4 months after gene transfer. These mice will be evaluated at 6.5 months after gene transfer (8 months of age) by echocardiography, electroretinogram, and auditory-evoked brainstem response to determine if gene transfer into adults can prevent the clinical manifestations of MPS I. These studies indicate that CTL responses can occur when using gene therapy to treat LSD. It is interesting to speculate that uptake of enzyme by antigen presenting cells via the mannose or mannose 6-phosphate receptor may potentiate antigen presentation by Class I MHC molecules, a hypothesis that is currently being tested. It may be necessary to modulate the immune system to achieve stable expression after gene transfer into older animals.

Hematopoietic stem cells (HSC) are important targets for gene therapy. Most protocols involve ex vivo modification, in which HSC are transduced in vitro and injected into the recipient. An in vivo delivery method might simplify HSC gene therapy. We previously demonstrated that iv injection of an amphotropic retroviral vector (RV) into newborn mice resulted in long-term expression from hepatocytes. The goal of this study was to determine if HSC were also transduced. After neonatal administration of 1 × 1010 transducing units/kg of RV, peripheral blood cells had ∼0.1 copy of RV per cell for up to 22 months. At 18 months, RV sequences were detected in T, B, and myeloid cells from bone marrow (BM). Unfractionated BM was transplanted into naive recipients after total body irradiation. Recipients maintained similar levels of the RV in their blood cells for 10 months, at which time RV sequences were present at the same integration site in all lineages of cells from BM. We conclude that neonatal iv injection of RV results in transduction of HSC in mice, which might be used for BM-directed gene therapy. Transduction of blood cells after liver-directed neonatal gene therapy might have adverse effects in patients, although no leukemias developed here.

The acute-phase response can result in decreased liver-specific functions and death as a result of liver failure. We show here that lipopolysaccharide (LPS), an endotoxin that induces the acute-phase response, results in a marked decrease in the major isoforms of the transcription factor, hepatocyte nuclear factor 4 alpha (HNF-4 alpha), in livers of rats. HNF-4 alpha is a nuclear receptor that is critical for the expression of several liver-specific genes. This decrease in HNF-4 alpha is primarily the result of a posttranscriptional mechanism, because mRNA levels are normal, and there are no major changes in the splicing patterns. This decrease was of functional significance, because expression of a gene that is highly dependent on HNF-4 alpha, HNF-1 alpha, was reduced. Interleukin-1 beta (IL-1 beta) is a cytokine whose levels are increased in vivo in response to LPS. IL-1 beta resulted in a decrease in HNF-4 alpha levels in HepG2 cells. This IL-1 beta-induced decrease was likely caused by degradation via the proteasome, because it was prevented by the addition of the proteasome inhibitor, MG132. We conclude that the decrease in HNF-4 alpha that occurs in vivo after the administration of LPS may be the result of IL-1 beta-induced degradation, and likely contributes to the liver insufficiency that occurs. IL-1 beta antagonists or proteasome inhibitors might increase HNF-4 alpha protein levels in the acute-phase response, which could result in increased liver function and survival.