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Mucopolysaccharidosis type I (MPS I) is a severe disease due to deficiency of the lysosomal hydrolase α-L-iduronidase (IDUA) and the subsequent accumulation of the glycosaminoglycans (GAG), leading to progressive, systemic disease and a shortened lifespan. Current treatment options consist of hematopoietic stem cell transplantation, which carries significant mortality and morbidity risk, and enzyme replacement therapy, which requires lifelong infusions of replacement enzyme; neither provides adequate therapy, even in combination. A novel in vivo genome-editing approach is described in the murine model of Hurler syndrome. A corrective copy of the IDUA gene is inserted at the albumin locus in hepatocytes, leading to sustained enzyme expression, secretion from the liver into circulation, and subsequent uptake systemically at levels sufficient for correction of metabolic disease (GAG substrate accumulation) and prevention of neurobehavioral deficits in MPS I mice. This study serves as a proof-of-concept for this platform-based approach that should be broadly applicable to the treatment of a wide array of monogenic diseases., In vivo genome editing following a single injection of ZFN and IDUA donor encoding AAV8 resulted in metabolic correction and neurological benefit in a murine model of MPS I. These results enable a currently open clinical trial to evaluate targeted genome engineering for MPS I in humans.

Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal disorder caused by deficiency of iduronate 2-sulfatase (IDS), leading to accumulation of glycosaminoglycans (GAGs) in tissues of affected individuals, progressive disease, and shortened lifespan. Currently available enzyme replacement therapy (ERT) requires lifelong infusions and does not provide neurologic benefit. We utilized a zinc finger nuclease (ZFN)-targeting system to mediate genome editing for insertion of the human IDS (hIDS) coding sequence into a “safe harbor” site, intron 1 of the albumin locus in hepatocytes of an MPS II mouse model. Three dose levels of recombinant AAV2/8 vectors encoding a pair of ZFNs and a hIDS cDNA donor were administered systemically in MPS II mice. Supraphysiological, vector dose-dependent levels of IDS enzyme were observed in the circulation and peripheral organs of ZFN+donor-treated mice. GAG contents were markedly reduced in tissues from all ZFN+donor-treated groups. Surprisingly, we also demonstrate that ZFN-mediated genome editing prevented the development of neurocognitive deficit in young MPS II mice (6–9 weeks old) treated at high vector dose levels. We conclude that this ZFN-based platform for expression of therapeutic proteins from the albumin locus is a promising approach for treatment of MPS II and other lysosomal diseases., AAV-mediated in vivo delivery of ZFN and IDS donor resulted in site-specific gene insertion and dose-dependent IDS expression in a mouse model of MPS II. These results support a currently open clinical trial, the first ever in vivo human genome editing study to be conducted.

Lentiviral vectors enter cells with high efficiency and deliver stable transduction through integration into host chromosomes, but their preference for integration within actively transcribing genes means that insertional mutagenesis following disruption of host proto-oncogenes is a recognized concern. We have addressed this problem by combining the efficient cell and nuclear entry properties of HIV-1–derived lentiviral vectors with the integration profile benefits of Sleeping Beauty (SB) transposase. Importantly, this integration enzyme does not exhibit a preference for integration within active genes. We generated integrase-deficient lentiviral vectors (IDLVs) to carry SB transposon and transposase expression cassettes. IDLVs were able to deliver transient transposase expression to target cells, and episomal lentiviral DNA was found to be a suitable substrate for integration via the SB pathway. The hybrid vector system allows genomic integration of a minimal promoter-transgene cassette flanked by short SB inverted repeats (IRs) but devoid of HIV-1 long terminal repeats (LTRs) or other virus-derived sequences. Importantly, integration site analysis revealed redirection toward a profile mimicking SB-plasmid integration and away from integration within transcriptionally active genes favored by integrase-proficient lentiviral vectors (ILVs).

Spliceosome-mediated RNA trans-splicing (SMaRT) is an emerging technology for the repair of defective pre-messenger RNA (pre-mRNA) molecules. It is especially useful in the treatment of genetic disorders involving large genes. Although viral vectors have been used for achieving long-lasting expression of trans-splicing molecules, the immunogenicity and suboptimal safety profiles associated with viral-based components could limit the widespread application of SMaRT in the repair of genetic defects. Here, we tested whether the non-viral Sleeping Beauty (SB) transposon system could mediate stable delivery of trans-splicing molecules designed to correct the genetic defect responsible for severe combined immune deficiency (SCID). This immunological disorder is caused by a point mutation within the 12.4 kilobase (kb) gene encoding the DNA protein kinase catalytic subunit (DNA-PKcs) and is associated with aberrant DNA repair, defective T- and B-cell production, and hypersensitivity to radiation-induced injury. Using a novel SB-based trans-splicing vector, we demonstrate stable mRNA correction, proper DNA-PKcs protein production, and conference of a radiation-resistant phenotype in a T-cell thymoma cell line and SCID multipotent adult progenitor cells (MAPCs). These results suggest that SB-based trans-splicing vectors should prove useful in facilitating the correction of endogenous mutated mRNA transcripts, including the DNA-PKcs defect present in SCID cells.

Mucopolysaccharidosis type II (MPS II; Hunter Syndrome) is an X-linked inherited lysosomal storage disease caused by deficiency of iduronate-2-sulfatase (IDS) and subsequent accumulation of glycosaminoglycans (GAGs) dermatan and heparan sulphate. Affected individuals exhibit a range in severity of manifestations such as organomegaly, skeletal dysplasias, cardiopulmonary obstruction, neurocognitive deficit, and shortened life expectancy. There is no cure for MPS II at the moment. Current standard of care is enzyme therapy (ELAPSRASE; idursulfase), which is used to manage disease progression. As hematopoetic stem cell transplantation (HSCT) has not shown neurologic benefit for MPS II, there is currently no clinical recourse for patients exhibiting neurologic manifestations of this disease, and new therapies are desperately needed.We have been developing the use of AAV9 vectors for delivery of the human IDS gene into the central nervous system of MPS II mice to restore IDS levels in the brain and prevent the emergence of neurocognitive deficits in the treated animals. A series of CBA-regulated vectors were generated that encode human IDS with or without the human sulfatase modifying factor-1 (SUMF-1), required for activation of the sulfatase active site. Intrathecal (IT) administration of these vectors into IDS positive mice (NOD.SCID and C57BL/6) did not result in a significant increase in IDS levels in the brains or in the organs of treated mice when compared to untreated animals. However, plasma showed higher enzyme levels in the intrathecally treated C57BL/6 mice than in untreated animals that persisted for at least 6 weeks post injection. There was no evidence for diminished activity that would be associated with immune response against human IDS protein. In contrast to the IT administered mice, MPS II mice administered AAV9-IDS vector intracerebroventricularly exhibited two- to four-fold higher levels of IDS enzyme activity in all portions of the brain in comparison with untreated, unaffected littermates. The ICV treated animals also showed lower levels of tissue GAG in the brain than untreated MPS II animals and similar levels as their wild-type littermates. IDS activity in the plasma of ICV treated mice was also significantly higher than that of wild type mice with no sign of immune response at least 6 weeks post administration. These results show the potential therapeutic benefit of AAV9-mediated IDS gene delivery to the CNS through the cerebrospinal fluid to address neurological manifestations of MPS II, Hunter syndrome.

Sleeping Beauty (SB) is a DNA transposon capable of mediating gene insertion and long-term expression in vertebrate cells when co-delivered with a source of transposase. In all previous reports of SB-mediated gene insertion in somatic cells, the transposase component has been provided by expression of a co-delivered DNA molecule that has the potential for integration into the host cell genome. Integration and continued expression of a gene encoding SB transposase could be problematic if it led to transposon re-mobilization and reintegration. We addressed this potential problem by supplying the transposase-encoding molecule in the form of mRNA. We show that transposase-encoding mRNA can effectively mediate transposition in vitro in HT1080 cells and in vivo in mouse liver following co-delivery with a recoverable transposon or with a luciferase transposon. We conclude that in vitro-transcribed mRNA can be used as an effective source of transposase for SB-mediated transposition in mammalian cells and tissues.

The Sleeping Beauty transposon system (SB) has been shown to mediate nonviral integration of expression constructs resulting in long-term gene expression in several mammalian targets. Often, however, it is difficult to discern long-term expression resulting from transposition vs nonhomologous chromosomal recombination or maintenance of plasmid DNA in an extrachromosomal form. We have designed a system to silence expression from nontransposed sequences, making it possible to determine more specifically the amount of expression resulting from transposition. A transposon plasmid, pT2F/Cage (carrying a murine erythropoietin (Epo) gene transcriptionally regulated by the ubiquitously expressed CAGS promoter), was engineered to contain LoxP sites positioned so as to interrupt expression upon Cre-mediated recombination. Upon transposition these sites become segregated, thus protecting the expression construct from Cre-mediated recombination and subsequent silencing. Interferon-inducible Mx1Cre mice were administered pT2F/Cage with or without transposase-encoding plasmid. At 2 to 4 weeks postinjection, in the absence of SB transposase, Cre induction reduced Epo expression to about 1% of that seen in the group that was administered transposase-encoding plasmid, which maintained Epo levels near those of the uninduced groups. Southern hybridization analysis and plasmid rescue of transfected tissue supported the efficient Cre-mediated silencing of nontransposed sequences. These results indicate a substantial level of DNA-mediated expression not associated with transposition, but which can be quantitatively distinguished from transposition by its sensitivity to Cre recombinase. The results also provide additional evidence for the effectiveness of the Sleeping Beauty transposon system as an in vivo DNA-mediated gene transfer strategy for achieving long-term expression.

The 2A-like sequences from members of the picornavirus family were utilized to construct a tricistronic vector bearing the human iduronidase (IDUA) gene along with the firefly luciferase and DsRed2 reporter genes. The 2A-like sequences mediate a cotranslational cleavage event resulting in the release of each individual protein product. Efficient cleavage was observed and all three proteins were functional in vitro and in vivo, allowing for supratherapeutic IDUA enzyme levels and the coexpression of luciferase and DsRed2 expression, which enabled us to track gene expression.

Previous studies have identified Sleeping Beauty transposons as efficient vectors for nonviral gene delivery in mammalian cells. However, studies demonstrating the usefulness of transposons as gene delivery vehicles into adult stem cells are lacking. Multipotent adult progenitor cells (MAPC) are nonhematopoietic stem cells with the capacity to form most, if not all, cell types of the body and as such hold great therapeutic potential. The whole-body biodistribution and persistence of MAPC are unknown, and such data would help direct clinical applications. We have nucleofected murine MAPC with two plasmid-based Sleeping Beauty transposons encoding the red fluorescent protein (DsRed2) and firefly luciferase. Transgenic euploid MAPC clones maintained their characteristic multilineage differentiation potential in vitro. DsRed2 and luciferase expression allowed for MAPC detection in vivo and in tissue sections. To confirm that transgenesis occurred by transposition into the genome of MAPC, we mapped Sleeping Beauty transposon integration sites in two MAPC clones using splinkerette PCR. This novel dual-reporter imaging approach based on the transgenesis of MAPC with Sleeping Beauty transposons sheds light on the homing patterns of MAPC and paves the way for quantification of MAPC engraftment in real time in vivo. ispartof: Molecular Therapy vol:12 issue:1 pages:42-48 ispartof: location:United States status: published

Murine models of lysosomal storage diseases provide an opportunity to evaluate the potential for gene therapy to prevent systemic manifestations of the disease. To determine the potential for treatment of mucopolysaccharidosis type I using a gene delivery approach, a recombinant adeno-associated virus (AAV) vector, vTRCA1, transducing the human iduronidase (IDUA) gene was constructed and 1 x 10(10) particles were injected intravenously into 1-day-old Idua(-/-) mice. High levels of IDUA activity were present in the plasma of vTRCA1-treated animals that persisted for the 5-month duration of the study, with heart and lung of this group demonstrating the highest tissue levels of gene transfer and enzyme activity overall. vTRCA1-treated Idua(-/-) animals with measurable plasma IDUA activity exhibited histopathological evidence of reduced lysosomal storage in a number of tissues and were normalized with respect to urinary GAG excretion, craniofacial bony parameters, and body weight. In an open field test, vTRCA1-treated Idua(-/-) animals exhibited a significant reduction in total squares covered and a trend toward normalization in rearing events and grooming time compared to control-treated Idua(-/-) animals. We conclude that AAV-mediated transduction of the IDUA gene in newborn Idua(-/-) mice was sufficient to have a major curative impact on several of the most important parameters of the disease.

Mucopolysaccharidosis type I (MPS I) is characterized by progressive neurodegeneration, and premature death (

Gene transfer to the lung could provide important new treatments for chronic and acquired lung diseases such as cystic fibrosis, α1-antitrypsin deficiency, emphysema, and cancer. DNA-mediated gene transfer to the lung has been previously demonstrated, but anticipated effectiveness has been limited by low gene transfer efficiencies and by transient expression of the transgene. Here, we combine plasmid-based gene transfer with the integrating capacity of the nonviral Sleeping Beauty (SB) transposon vector system to mediate gene insertion and long-term gene expression in mouse lung. We observed transgene expression after 24 h in lungs of all animals injected with the luciferase transposon (pT/L), but expression for up to 3 months required codelivery of a plasmid encoding the Sleeping Beauty transposase. We also observed long-term expression in pT/L-injected animals transgenic for SB transposase. Transgene expression was localized to the alveolar region of the lung, with transfection including mainly type II pneumocytes. We used a linker-mediated PCR technique to recover transposon flanking sequences, demonstrating transposition of pT/L into mouse chromosomal DNA of the lung.

Mutations in DCLRE1C/Artemis, a DNA repair gene, cause T-B-NK+ SCID by preventing V(D)J recombination in T and B cell progenitors and also confer heightened sensitivity to irradiation and alkylator chemotherapy. SCID newborn screening identifies Artemis-deficient SCID (ART-SCID) early in life and while allogeneic hematopoietic cell transplantation can cure ART-SCID, preparative regimens for conditioning are poorly tolerated. Without alkylating chemotherapy patients may have graft failure while toxic effects of chemotherapy include increased mortality, short stature and abnormal dental development. Thus, building on experience with X-linked and ADA deficient SCID, we found addition of a normal Artemis gene to hematopoietic stem cells (HSC) an attractive strategy to treat ART-SCID.Since overexpression of Artemis protein causes cellular cytotoxicity, a lentivirus vector with human Artemis cDNA and its endogenous promoter (Apro-hART) was produced and used to transduce fibroblasts from ART-SCID patients and controls. Apro-hART transduced ART-SCID fibroblasts showed correction of radiosensitivity by enumeration of foci of DNA damage and proliferation assays. Radiosensitivity of cells lacking the DNA repair enzyme Ligase-4 was not corrected. View Large Image | Download PowerPoint SlideMobilized peripheral blood CD34+ cells from an ART-SCID patient, incapable of differentiation into T and B cells, were transduced with Apro-hART or GFP lentivirus and cultured on OP9 cells or injected into irradiated newborn NSG mice. OP9 cocultures and blood and spleen cells from NSG mice showed that Apro-hART-corrected, but not GFP-transduced, ART-SCID CD34+ cells differentiated into B cells and T and B cells, respectively, as did GFP-transduced control CD34+ cells. Lymphocyte maturation was proven by lineage specific surface markers and measures of V(D)J diversity and recombination, T cell receptor Vbeta spectratyping and Kappa chain receptor excision circles (KRECs), respectively. View Large Image | Download PowerPoint SlideColony forming assays with transduced cells revealed transduction efficiency of 28.6%, a mean vector copy number of 3/cell and a diverse profile of lentivirus integration sites.This successful gene correction and restoration of Artemis function in fibroblasts and HSCs from ART-SCID patients supports institution of a clinical trial of gene addition therapy for ART-SCID.

The non-viral integrating vector the Sleeping Beauty (SB) transposon system is efficient in treating systemic monogenic diseases in mice including mucopolysaccharidosis (MPS) types I and VII caused by α-iduronidase (IDUA) and β-glucuronidase (GUSB) deficiency, respectively. More recently we have used modified approaches of the hydrodynamic procedure to deliver therapeutic transposons to dog liver. Reproducible delivery and transposition in dogs are about 1% the levels in mice. Using a transgenic canine reporter secreted alkaline phosphatase (cSEAP), in the absence of immune suppression we can detect transgenic protein for up to six weeks post infusion using catheter-mediated hydrodynamic delivery. A proof-of-principle immunomodulation using GdCl3 to block macrophages in liver and spleen prolonged the presence of the cSEAP protein in circulation from 6 weeks to up to at least 5 months after a single vector infusion. We achieved stabilized activity in one dog at about 2-fold of baseline values. Durability of cSEAP in serum was inversely correlated with transient increase of liver enzymes ALT and AST in response to the vector delivery procedure, pointing to the deleterious effect of hepatocellular toxicity on transgene maintenance. However, GdCl3 immunomodulation was ineffective for repeat vector infusions, suggesting a possibility of an alternative, more potent immunosuppression regimen. Evidence of transposition was obtained with the most efficient transposase SB100X but not with SB11. For transgenic IDUA and GUSB, therapeutic activity in serum peaked at 50-350% of wild-type at 2-4 days post-treatment, but lasted only a few days. The differences in levels and duration of detection of cSEAP in the blood compared to those of IDUA and GUSB may be in part due to the facilitated uptake of lysosomal enzymes into cells compared to cSEAP. Longer endurance of transgenic proteins at therapeutic levels may be possible in SB-treated dogs using alternative immunosuppressive regimens.

Hunter syndrome (Mucopolysaccharidosis Type II, MPS II) is a rare X-linked lysosomal disorder caused by lack of functional iduronate-2 sulfatase (IDS) enzyme and subsequent accumulation of glycosaminoglycans (GAG) in affected individuals. Manifestations include skeleton dysplasia, splenohepatomegaly, cardiopulmonary obstruction, and shortened life expectancy. In severe cases there is also neurologic impairment. Enzyme replacement therapy (ERT) is currently the only FDA-approved treatment to manage disease progression; however, ERT does not affect neurological aspects of the disease and requires that patients receive long and costly infusions of replacement factor on a frequent basis. We have developed a zinc-finger nuclease (ZFN) approach to insert the human IDS (hIDS) coding sequence into the albumin locus using AAV2/8 vectors. In this study IDS-deficient MPS II mice (n= 8-13 per group, age 7-8 weeks) were treated by intravenous infusion of a mixture of ZFN-encoding AAV vectors along with an AAV vector encoding the hIDS partial cDNA flanked by albumin sequence homology arms at three different vector doses. Wild-type littermates, untreated MPS II mice, and MPS II animals infused only with the hIDS donor vector (without ZFN-encoding vectors) were included as controls. Successful insertion of the hIDS coding sequence will result in hIDS expression regulated by the endogenous albumin promoter. Plasma and tissue IDS activities as well as urine and tissue GAG contents were monitored throughout the study to evaluate the effectiveness of the treatment. Sufficient animals were maintained for neurobehavioral testing at four-months post-injection to determine whether the treatment is neurologically beneficial. We found that IDS activities in the plasma of the treated groups were 10- to 100-fold higher than wild-type and stably expressed through the entire study duration in a dose-dependent fashion, while only very low levels of IDS activity were found in the animals infused with hIDS donor vector alone. At 4 weeks post-treatment IDS activities in peripheral tissues ranged from 1% to 200% wild-type in a dose-dependent fashion, while in the hIDS donor-only group enzyme activity was not detected in any tissue except liver (10% that of wild-type). We observed up to 2% of the wild-type IDS activity in the brains of animals administered the complete set of AAV vectors, while no IDS activity was observed in the brains of animals infused with the IDS donor vector alone. Urine GAGs were reduced in all of the ZFN + Donor treatment groups regardless of the vector dose. Tissue GAGs in the treatment groups were also decreased, but GAG content in the brain was not different from untreated MPS II litter mates at the initial analysis conducted four weeks post-treatment. No tissue GAG reduction was observed in animals infused with hIDS donor vector alone. Before conclusion of this study, animals from all six groups will be tested in the Barnes Maze as neurobehavioral assessment to determine the neurological effect of targeted hIDS expression in the liver. These results together with hIDS expression and GAG level data from final necropsy tissues will be presented. These results demonstrate that ZFN can be effectively used to mediate in vivo insertion of the hIDS coding sequence into the albumin locus with resultant stable and high-level hIDS enzyme expression and metabolic correction in MPS II.

Mucopolysaccharidosis type II (MPS II; Hunter Syndrome) is an X-linked recessive inherited lysosomal storage disease caused by deficiency of iduronate-2-sulfatase (IDS) and subsequent accumulation of glycosaminoglycans (GAGs) dermatan and heparan sulphate. Affected individuals exhibit a range in severity of manifestations such as organomegaly, skeletal dysplasias, cardiopulmonary obstruction, neurocognitive deficit, and shortened life expectancy. There is no cure for MPS II at the moment. Current standard of care is enzyme replacement therapy (ELAPSRASE; idursulfase), which is used to manage disease progression. However, enzyme replacement therapy (ERT) does not show neurologic improvement. As hematopoetic stem cell transplantation (HSCT) has not shown neurologic benefit for MPS II, there is currently no clinical recourse for patients exhibiting neurologic manifestations of this disease, and new therapies are desperately needed. We have been developing the use of AAV9 vectors for delivering the human IDS coding sequence (AAV9.hIDS) into the central nervous system of MPS II mice to restore IDS levels in the brain and prevent the emergence of neurocognitive deficits in the treated animals. A series of CMV-enhancer, beta actin-regulated vectors were generated that encode human IDS with or without the human sulfatase modifying factor-1 (SUMF-1), required for activation of the sulfatase active site. Three routes of administration; Intrathecal (IT), Intracerebroventricular (ICV) and Intravenous (IV) were used in these experiments. We found no significant difference in the enzyme level between mice that were treated with AAV9 vector transducing hIDS alone and mice that were treated with AAV9 vector encoding human IDS and SUMF-1, regardless of the route of administration. IT-administrated NOD. SCID (IDS Y+) and C57BL/6 (IDS Y+) did not show elevated IDS activity in the brain and spinal cord when compared to untreated animals, while plasma showed ten-fold higher (NOD. SCID) and 150-fold higher (C57BL/6) levels than untreated animals. IDS-deficient mice intravenously administered AAV9-hIDS exhibited IDS activities in all organs that were comparable to wild type. Moreover, the plasma of IV injected animals showed enzyme activity that was 100-fold higher than wild type. IDS-deficient mice administered AAV9-hIDUA intracerebroventricularly showed IDS activities comparable to wild type in most areas of the brain and peripheral tissues, while some portions of the brain showed two- to four-fold higher activity than wild type. Furthermore, IDS activity in plasma was 200-fold higher than wild type. Surprisingly, IDS enzyme activity in the plasma of all administrated animals showed persistence for at least 12 weeks post injection; therefore, IDS enzyme was not immunogenic at least on the C57BL/6 murine background. We also conducted additional neurobehavioral testing using the Barnes maze to differentiate neurocognitive deficits of untreated MPS II animals from that of wild type littermates. We found that the learning capability of affected animals is distinctively slower than that observed in littermates. Thus, Barnes maze will be used to address the benefit of these therapies in the MPS II murine model in future experiments. These results indicate potential of therapeutic benefit of AAV9 mediated human IDS gene transfer to the CNS to prevent neurologic deficiency in the MPS II.

Lysosomal storage diseases (LSDs) represent a group of inherited metabolic disorders associated with mutations in genes encoding lysosomal enzymes leading to systemic accumulation of toxic storage materials. Manifestations include organomegaly, skeletal dysplasias, cardiopulmonary obstruction, and severe neurologic impairment, often leading to death by age 10. Current LSD therapies include enzyme replacement therapy, substrate reduction therapy and hematopoietic stem cell transplant (HSCT). However, all of these therapies are expensive, incompletely effective, and HSCT is associated with significant risk of morbidity and mortality. Due to the unmet need genome editing strategies are proposed to permanently modify patient cells by genetically complementing the LSD defect. This is achieved by utilizing engineered zinc finger nucleases (ZFNs) to introduce a DNA cut at the target locus, mediating integration of a therapeutic LSD donor cDNA. To ensure long-term expression of the transgenes in vivo we target the albumin locus as a “safe harbor” in hepatocytes via co-delivery of the albumin ZFNs and LSD donors by adeno-associated virus (AAV). This system exploits the high transcriptional activity of the native albumin enhancer/promoter; uses stably modified hepatocytes to potentially allow long-term expression of the inserted transgene; and utilizes an endogenous promoter obviating this requirement in the AAV payload. We have previously exploited AAV-mediated in vivo targeting of the murine albumin “safe harbor” locus for the synthesis of therapeutic levels of FVIII and FIX to overcome the clotting defect in hemophilic mice. Using the same approach, we co-delivered mouse albumin ZFNs with donor constructs encoding either human iduronate-2-sulfatase (IDS) deficient in Hunter syndrome, α-L-iduronidase (IDUA) for Hurler syndrome, or Glucocerebrosidase (GBA) for Gaucher disease via AAV in WT mice. We show stable integration of the LSD donors at the albumin locus, resulting in liver-specific expression and secretion of these proteins into plasma. This led to a 4-fold (GBA), 10-fold (IDUA) or 100-fold (IDS) increase in enzymatic activity in the plasma, demonstrating that the secreted proteins are functional. Importantly, increased activity was also detected in secondary tissues (spleen) showing efficient uptake and activity in distal tissues. Moreover, preliminary data in MPSI and MPSII mice suggests these levels of IDS and IDUA expression may lead to correction of the enzyme deficiency. IDS, IDUA and GBA expression remained stable throughout the study (up to 2 months), while expression of FIX has been stable for >1 yr suggesting this process results in long-term protein expression. In summary, our data provide proof of concept for ZFN-mediated targeting of the albumin locus in hepatocytes as an in vivo protein replacement platform to express different proteins associated with lysosomal storage diseases.

Hemophilia A and hemophilia B are X-linked, genetic disorders that are caused by defective or deficient coagulation factor VIII and IX, respectively, resulting in the inability to form blood clots and sustained bleeding after trauma or injury. Recombinant clotting factor protein is currently used to treat hemophilia at a high cost per patient. As a therapeutic approach for hemophilia, gene transfer has the potential to provide more consistent levels of circulating clotting factor over an extended period of time for more cost-effective treatment. Moreover, only modest levels of FVIII or FIX expression (2%-5% of normal) can improve clinical outcomes. We are using the Sleeping Beauty (SB) transposon system to engineer autologous human B cells for secretion of clotting factors as a cellular therapy for hemophilia. An in vitro system for expansion and differentiation of memory B cells into plasma cells has been developed. Plasma cells are suitable for sustained delivery of FVIII or FIX, since they secrete high levels of protein and may survive for years in vivo. For human FIX expression, we assembled an SB transposon with the human FIX coding sequence (codon-optimized with R338L mutation for enhanced potency) regulated by the CAGS promoter. Elevated levels of hFIX in cultures of B lymphoblastoid cells required co-electroporation of the hFIX transposon along with an SB transposase encoding plasmid, demonstrating the role of transposition in achieving extended hFIX expression. There have been remarkable advances recently in the treatment of hemophilia B by systemic AAV8-hFIX administration, but similar treatment of hemophilia A presents a significant challenge due to the size of the FVIII-encoding sequence and the complexity of the protein. We previously demonstrated B-domain deleted hFVIII expression and correction of clotting dysfunction in FVIII deficient mice by hydrodynamic delivery using the SB transposon system (Ohlfest et al, Blood 105: 2691, 2005). Current studies are focused on identifying conditions for effective hFVIII transposon delivery and long term expression in primary human B cells after Sleeping Beauty-mediated transposition. Results from these studies will be applicable to the development of a clinical protocol for treatment of human hemophilia by infusion of B cells genetically engineered using the Sleeping Beauty transposon system.

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive storage disease caused by deficiency of alpha-L-iduronidase (IDUA), resulting in accumulation of glycosaminoglycans (GAGs). In the severe form of the disease (Hurler syndrome), death results by age 10. Current treatments for this disease include hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT). However, ERT is ineffective in treating CNS disease due to the inability of lysosomal enzymes to traverse the blood-brain barrier, and while there is neurologic benefit to HSCT the procedure is associated with significant morbidity and mortality.We have taken a novel approach to treat neurologic disease associated with Hurler syndrome, using intranasal administration of an IDUA-encoding AAV9 vector. A CAGS regulated AAV9-IDUA vector was infused intranasally into adult mice (2-3 months of age) that had been immunotolerized at birth with Aldurazyme to prevent anti-IDUA immune response. Mice sacrificed at 3 months post-infusion exhibited IDUA enzyme activity levels that were 100-fold that of wild type in the olfactory bulb, with wild type levels of enzyme restored in all other parts of the brain. Intranasal treatment with AAV9-IDUA also resulted in clearance of tissue GAG storage materials in all parts of the brain. QPCR analysis of vector genomes indicated only background levels in all portions of the brain. There was strong IDUA immunofluorescence staining of tissue sections observed in the nasal epithelium and olfactory bulb but there was no evidence for the presence of transduced cells in other portions of the brain. This indicates that clearing of storage materials most likely occurred as a result of enzyme diffusion from the olfactory bulb and the nasal epithelium into deeper areas of the brain. At 6 months of age, intranasally treated animals along with age-matched heterozygote and IDUA-deficient control animals were subjected to neurocognitive testing using the Barnes maze. Unaffected heterozygote animals exhibited improved performance in this test while MPS I mice displayed a deficit in locating the escape. Remarkably, MPS I mice treated intranasally with AAV9-IDUA exhibited behavior similar to the heterozygote controls, demonstrating prevention of the neurocognitive deficit seen in the untreated MPS I animals. There was no significant difference between heterozygote animals and treated animals, while latency to escape was significantly different between these two groups and MPS I deficient animals (P

Many applications of gene therapy require long-lasting expression. In mice, hydrodynamic delivery directs DNA to the liver where when integrated expression can last the lifetime of the animal. Because this form of delivery will be invasive in larger animals, we have been seeking alternative methods of delivery, including condensing DNA with polyethelenimine (PEI). PEI has been reported to deliver genes to the lung with expression lasting for up to 3 months (Belur et al, Molecular Therapy 8: 501|[ndash]|507; 2003). However, using standard hydrodynamic delivery as a |[ldquo]|gold standard|[rdquo]| for achievable levels of gene expression in mice, we have found that simple PEI condensation results in expression of delivered genes at levels that are 10 to more than 100-fold lower than with hydrodynamic delivery. Following injection into the tail vein of PEI-complexed plasmids containing an SB transposon with a luciferase gene, DNA expression was most pronounced in the lung one day post-injection. By 7 days post-injection, expression was most evident in the liver rather than the lung as visualized with a Xenogen|[trade]| in vivo imaging system. JetPEI|[trade]| at N/P ratios of 7 and 9 led to expression levels that ranged from 1|[ndash]|22 |[times]| 106 photons/second (p/s) in the lung 1-day post-injection. However, by day-7 the ranges were from 2|[ndash]|80 |[times]| 104 to 2|[ndash]|9 |[times]| 104 p/s for N/P ratios of 7 and 9, respectively. Because particle sizes should be 100 nm or less for uptake by endocytosis, we sized our PEI-plasmid particles by dynamic laser light scattering. Particles ranged in size from 80|[ndash]|100 nm in 5% dextrose at an N/P of 7 with JetPEI|[trade]|. Because uncomplexed, free PEI is cytotoxic to cells, we hypothesize that the removal of free PEI will permit higher N/P ratios, which might provide increased levels of gene delivery and expression. Consequently, we are testing methods for removal of free PEI so that PEI transposon complexes prepared at higher N/P ratios can be tested for improved levels of long-term expression and animal survival after in vivo delivery.

The Sleeping Beauty (SB) transposon system combines the advantages of non-viral plasmid based gene transfer with an integrative ability, thus leading to long term expression of the transgene. Our goal is to use the SB transposon system to deliver antiangiogenic and immunostimulatory genes, and to test their antitumor effectiveness in an animal model of colorectal cancer metastases to the liver.

Messenger RNA (mRNA) has several advantages over DNA for gene transfer and expression, including the lack of any requirement for nuclear localization or transcription and the nearly negligible possibility of genomic integration of the delivered sequence. However, the labile nature of mRNA and its capacity to elicit innate immune responses are important limitations to its in vivo application. Now, an article by Kormann et al.1 published in Nature Biotechnology addresses these drawbacks and provides new hope for the potential use of mRNA as a source of therapeutic gene product in vivo.

Sleeping Beauty (SB) is a DNA transposon capable of mediating chromosomal integration and stable expression in vertebrate cells when co-delivered with a source of transposase. In all pre-clinical reports where SB-mediated gene insertion in somatic cells has been used to correct mouse models of human disease, the transposase component has been provided as a co-delivered DNA molecule that has the potential for integration into the host cell genome. Integration and continued expression of a gene encoding SB transposase could be problematic if it led to remobilization and reintegration of transposons. Such continued expression of transposase is a key safety concern in development of the SB transposon system for clinical applications. As an alternate source of transposase, we have previously shown that in vitro transcribed transposase-encoding messenger RNA (mRNA) can effectively mediate transposon insertion both in vitro and in mouse liver (Wilber et al., Molecular Therapy, 2005, in press). Here, we test the use of transposase- encoding mRNA plus transposon DNA for gene therapy of hereditary tyrosinemia type I by first evaluating several parameters for systemic delivery and expression of mRNA in mice. We also introduce a method to quantitatively track repopulating liver cells by in vivo bioluminescence imaging after co-delivery of a DNA or RNA source of transposase with a bi-functional transposon encoding both mouse fumaryl acetoacetate hydrolase (FAH) and firefly luciferase (luc) genes in FAH deficient mice by rapid, high-volume injection into the tail vein. Liver repopulation was quantitatively monitored over time by increasing luc activity, measured as light emitted from the liver. Using this method, we determined that supplying SB transposase in the form of mRNA results in selective repopulation of corrected hepatocytes with stable co-expression of both FAH and luc. Plasma taken from animals 5 months after co-infusion with transposase mRNA contained levels of succinylacetone (the clinical determinant of tyrosinemia) that were nearly normalized. Amino acid levels were also normalized, suggesting normal liver metabolism of catabolized protein products (including urea and glucose). We further demonstrated the stability of integration by transplanting hepatocytes (250,000) into FAH deficient recipient mice. All transplanted animals survived NTBC withdrawal and gained weight consistently over a period of 90 days and demonstrated stable expression of luc. In summary, we demonstrate for the first time that transposase-encoding mRNA can be used to mediate non-viral gene therapy resulting in complete phenotypic correction that is stable and not associated with any incidents of cellular transformation.

Top of pageAbstract The purpose of this project was to evaluate the Sleeping Beauty (SB) transposon for delivery of the human |[alpha]|-L-iduronidase ( IDUA) gene to chromosomes in the livers of IDUA-deficient mice for long- term expression and correction of MPS type I. We chose the hydrodynamics-based DNA injection, which targets mainly the liver. We constructed SB transposon, plasmid-based vectors and injected them into idua-/-mice. Because our goal was in part to determine the efficacy of transposition as a way of providing long-term expression of IDUA protein, control groups of MPS I mice did not get the transposase. Blood samples for plasma isolation were collected 1 day after treatment and once every 2 weeks thereafter. Plasma IDUA activities reached >100-fold of wild type levels on day 1 following treatment, but were essentially gone in all mice 4 weeks following gene delivery. IDUA activity was not detected in the livers of these mice 3 months after plasmid administration. We examined the duration of the transposon-delivered human IDUA DNA maintenance and enzyme activity over 6 months in the liver of wild type (WT) C57Bl/6 mice that express IDUA. As in the MPS I mice, plasma and liver IDUA activity reached supra-normal levels on day 1 and remained at this level for the first week, but still dropped dramatically within 2 weeks and by 4 weeks were indistinguishable from background. Using DNA PCR, we found that the maintenance of the IDUA transgene mirrored the IDUA activity time-line. Even though transposition was confirmed by an excision assay, the PCR product was detectable only for the first two weeks, but not thereafter. It appears that cells that express the IDUA gene are cleared from the liver of treated mice by 4 weeks following injection, which suggests induction of an immune response either to the therapeutic protein or/and the cells that express the therapeutic gene. This hypothesis is supported by our observations that in all cyclophosphamide immune-suppressed MPS I mice, the initial 1-day plasma activity levels dropped more than 100-fold by 2 weeks, but then persisted at 20-500% WT activity in some mice. IDUA levels were stable (up to 5-fold higher than in the WT mice) in SB-transposase-treated MPS I mice whereas without SB, IDUA levels declined to near-background over 3 months. These persistent IDUA activity levels were sufficient to reduce the number and size of pathologic inclusions in the liver seen by toluidine blue staining. Secondary elevation of murine |[beta]|-glucuronidase, another lysosomal enzyme, was reduced in the liver, spleen and kidney and correlated with degree of reduction of lysosomal pathology in these organs. Thus, with immune suppression, a single dose of the SB transposon system can result in partial to complete biochemical correction of IDUA activity in the livers of treated adult MPS I mice.

We are moving forward with development of our proprietary Sleeping Beauty Transposon (SBT) system for the treatment of Fanconi anemia (FA) via ex vivo electroporation of hematopoietic stem cells. FA is an inherited recessive disorder caused by deficiency in one of a number of genes whose products form a complex involved in DNA repair. The primary hematological hallmarks of the disease are aplastic anemia, bone marrow failure, and increased susceptibility to leukemias thought to be caused by defective cellular mechanisms for DNA repair. Allogeneic bone marrow transplantation is currently the only curative treatment for these aspects of the disease and despite improvements in clinical protocols over the years difficulties with allotransplants for FA still persist. The primary goal of this study is to evaluate the potential of SBT vectors carrying the FANCC gene to effectaffect transposition and establish long-term expression in FANCC-deficient cell populations after introduction by electroporation. Additionally, we show that integration of FANCC transposons can correct the sensitivity of lymphoblastoid cell lines (LCL) derived from FANCC patients to DNA damaging agents such as mytomycin C. We constructed a transposon containing a PGK promoter to regulate transcription of the normal human FANCC cDNA sequence. The vector containing the transposon also includes a ubiquitin promoter regulating expression of the SB transposase gene, encoded outside of the transposon. The SBT-FANCC vector was introduced via electroporation into FANCC deficient and normal control LCLs. Long-term maintenance of FANCC complementation in the FANCC-deficient cells was observed as a sustained reduction in sensitivity to mitomycin C similar to normal controls. In addition, the isolation and molecular characterization of clonal populations of these FANCC-complemented cells demonstrated FANCC transposon integration into the genome by SB mediated transposition. Preliminary results using SBT vectors in K562 cells suggest that a transposase provided on a separate plasmid may increase the efficiency of transposition. As a result, studies are also underway to characterize the efficacy of the SBT-FANCC vector described above compared to a similar vector in which the transposase is supplied either by a separate plasmid or by a SB-encoding mRNA. These studies demonstrate molecular feasibility of the SB transposon system in combination with electroporation-based cell loading technology for genetic therapy of Fanconi anemia.

Deficiency of fumaryl acetoacetate hydrolase (FAH) in hereditary tyrosinemia type I results in oxidative damage to hepatocytes. Treatment includes restriction of dietary intake of tyrosine and administration of 2-(2-nitro-4-trifluoromethylbenzyol)-1,3-cyclohexanedione (NTBC), which inhibits an enzyme upstream of FAH in tyrosine catabolism. FAH-/- knockout mice recapitulate the human disorder and require NTBC for survival. The mice have been used as a model system to study liver repopulation by wild-type hepatocytes or by FAH mutant hepatocytes that were corrected with wild-type FAH via viral or Sleeping Beauty (SB)-mediated gene transfer. Here we introduce a method to quantitatively track repopulating liver cells in vivo by bioluminescent imaging. We assembled a SB transposon plasmid with the strong CAGGS promoter regulating murine FAH expression. To the region 3' of the FAH coding sequence we attached an internal ribosome entry site (IRES) sequence and the luciferase transgene such that FAH and luciferase are translated from the same mRNA. Also, a SB transposase gene regulated by the PGK promoter was inserted into the plasmid outside of the transposon. For gene delivery to hepatocytes, we used rapid, high-volume injection into the tail vein of FAH-/- mice. Liver repopulation by SB-transposed cells was initiated by withdrawal of NTBC, which induces tyrosinemia. Six weeks after DNA injection the mice are steadily increasing in weight without NTBC, suggesting that the metabolic defect has been corrected. We also studied these mice for expression of the luciferase reporter gene contained on the same transposon sequence as the FAH gene. At various times after injection of tranposon DNA, the mice were injected i.p. with luciferin, and luciferase expression was visualized and quantified in vivo using the IVIS|[reg]| imaging system (Xenogen Corp.). We found that luciferase expression increased by 10-fold during two weeks of selective repopulation. We have engineered additional transposon constructs to link FAH expression with that of any transgene of interest, such as a mutant alpha1-antitrypsin gene. One potential application of this method is to generate liver-transgenic models without the necessity of germline transgenesis. In conclusion, we have developed a method to quantitatively track repopulating liver cells in vivo using luciferase as a reporter, and demonstrate that FAH-/- mice can be used for transgenesis of the adult mouse liver.

The Sleeping Beauty (SB) transposon system has been demonstrated to mediate gene insertion and long-term expression, suggesting the potential application of this plasmid based system for non-viral gene therapy. All reported studies of SB-mediated gene transfer to somatic cells have supplied the transposase by expression of co-injected plasmid DNA. Although successful for both in vitro and in vivo transposition, there is a lack of control with respect to the transient and long-term levels of transposase expressed in a target cell. There is also the potential for genomic integration of the transposase encoding DNA, resulting in sustained transposase expression and potential destabilizing effects on the integrated transgene or host genome integrity. We address these problems by supplying the transposase-encoding molecule in the form of mRNA. Rapid, high pressure injection of m7G-capped luciferase (Luc) mRNA through the tail vein of mice resulted in efficient luciferase expression with respect to a DNA element where the Caggs promoter controlled luciferase transcription. Using this system as a model for efficient gene transfer in the liver, we have taken steps to optimize delivery/expression of the mRNA by generating constructs to promote stability and translation. Real-time whole body in vivo imaging of Luc mRNA translation was conducted to measure the effect of an IRES and UTR sequences on luciferase expression. These studies determined that incorporation of 5' and 3' UTR elements from the Xenopus -globin gene were essential to achieve peak luciferase expression levels from an otherwise unprotected transcript. Co-delivery of these mRNAs with an inhibitor of or a substrate for endogenous RNases provided luciferase expression sufficient to mimic levels achieved using promoters known to provide optimum transposase expression for in vivo transposition. Using this genetic arrangement, we have co-delivered SB transposase mRNA with a transposon encoding the Luc gene (driven by the Caggs promoter) to the liver of transgenic mice in which transcription of Cre recombinase is under the control of an interferon response element. To distinguish between plasmid-mediated expression and expression mediated by transposition, we have engineered this transposon encoding plasmid with lox-P sites positioned such that Cre-mediated recombination excises a sequence which includes the Caggs promoter, unless it has been segregated away from the plasmid by the SB transposase (Score et al., Mol. Ther. 7: S9, 2003). We found that pIpC induction of Cre recombinase caused a 1600-fold reduction in luciferase expression in animals infused with the transposon alone. While co-delivery of transposase mRNA resulted in a moderate degree of inadvertent Cre induction, we observed only a 45-fold reduction in luciferase expression following analogous pIpC administration. This remaining luciferase expression is most likely attributable to SB mediated transposition. These results provide the first evidence for use of SB transposase mRNA to mediate transposition and long-term expression in an in vivo setting.