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Definitions

• the nucleic acid sequence may comprise the mature hOTC of SEQ ID NO: 1
• the engineered hOTC nucleic acid sequence is less than 80% identical to the wild-type hOTC sequence over the mature sequence or full-length hOTC of the wild-type sequence (e.g., SEQ ID NO: 1) and expresses a functional hOTCase.
• the synthetic hOTC nucleic acid sequence comprises at least the mature hOTC of SEQ ID NO: 5 or a nucleic acid sequence at least about 96 to about 99.9 % identical thereto or a nucleic acid sequence comprising at least the mature hOTC of SEQ ID NO: 9 or a nucleic acid sequence at least about 96 to about 99.9 % identical thereto.
• the invention provides a viral vector comprising a hOTC gene encoding a chimeric ornithine transcarbamylase which comprises mature human ornithine transcarbamylase with a heterologous transit peptide, wherein the coding sequence from the mature human ornithine transcarbamylase is selected from the nucleic acid sequence comprising at least the mature hOTC of SEQ ID NO: 3, 4, 5, 8 or 9.
• a viral vector comprising a nucleic acid sequence encoding functional human ornithine transcarbamylase in preventing and/or treating hepatocellular carcinoma in a subject having OTCD.
• the subject is a human patient.
• the subject is heterozygous for OTCD and may exhibits late onset of symptoms.
• FIGs. 5A - 5C provides an alignment of the cDNA sequences of the wild- type hOTC, and five engineered sequences, GS [SEQ ID NO: 3], LW3 [SEQ ID NO:4], LW4 [SEQ ID NO: 5], LW5 [SEQ ID NO: 8] and LW6 [SEQ ID NO:9].
• the aligned sequences contain a Kozak sequence (first 14 nucleotides of LW3 and LW4) and a restriction enzyme site (following termination codon for LW3 and LW4), which are not part of the open reading frame.
• FIG. 1A For comparison purposes, a wild-type human OTC cDNA sequence is illustrated in FIG. 1A. This sequence encodes the human ornithine transcarbamylase of the amino acid sequence of FIGS. 1A - 1C. This same amino acid sequence is encoded by the engineered hOTC genes of FIGS. 2A - FIG. 5.
• RNA sequences and sequences which are 95% to 99%, or about 97%, or about 98% identical to one or more of these sequences are encompassed within the scope of this invention.
• Methods for aligning and determining RNA identity are known the art and include published and publically available web-based or commercially available databases and services. See, e.g., LocARNA, CARNA, as well as other programs identified elsewhere therein.
• sequence identity refers to the residues in the two sequences which are the same when aligned for correspondence.
• the length of sequence identity comparison may be over the full-length of the genome, the full-length of a gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides, is desired. However, identity among smaller fragments, e.g. of at least about nine nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides, may also be desired.
• Percent identity may be readily determined for amino acid sequences over the full-length of a protein, polypeptide, about 32 amino acids, about 330 amino acids, or a peptide fragment thereof or the corresponding nucleic acid sequence coding sequences.
• a suitable amino acid fragment may be at least about 8 amino acids in length, and may be up to about 700 amino acids.
• identity is determined in reference to “aligned” sequences.
• alignd sequences or alignments refer to multiple nucleic acid sequences or protein (amino acids) sequences, often containing corrections for missing or additional bases or amino acids as compared to a reference sequence.
• nucleotide sequence identity can be measured using FastaTM, a program in GCG Version 6.1.
• FastaTM provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. For instance, percent sequence identity between nucleic acid sequences can be determined using FastaTM with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in GCG Version 6.1, herein incorporated by reference.
• 5' ITR the hOTC coding sequences and any regulatory sequences
• AAV 3' ITR A shortened version of the 5 ' ITR termed AITR, has been described in which the D-sequence and terminal resolution site (trs) are deleted. In other embodiments, the full-length AAV 5 ' and 3 ' ITRs are used.
• sc in this context refers to self-complementary.
• Self- complementary AAV refers a construct in which a coding region carried by a recombinant AAV nucleic acid sequence has been designed to form an intra-molecular double-stranded DNA template.
• dsDNA double stranded DNA
• control sequences are "operably linked" to the hOTC gene sequences.
• operably linked refers to both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
• AAV viral vectors are well suited for delivery of the hOTC expression sequences described herein.
• Such AAV vectors may contain ITRs which are from the same AAV source as the capsid.
• the AAV ITRs may be from a different AAV source than that which supplies the capsid.
• AAV AAV9
• AAVl AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9
• others such as, e.g., those described in WO 2003/042397; WO 2005/033321, WO 2006/110689; US 7588772 B2, which are incorporated by reference herein] may be used in human subjects.
• a single promoter may direct expression of a single R A that encodes two or three or more genes, and translation of the downstream genes are driven by IRES sequences.
• a single promoter may direct expression of an RNA that contains, in a single open reading frame (ORF), two or three or more genes separated from one another by sequences encoding a self-cleavage peptide (e.g., T2A) or a protease recognition site (e.g., furin).
• ORF open reading frame
• a "replication-defective virus” or “viral vector” refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, where any viral genomic sequences also packaged within the viral capsid or envelope are replication-deficient; i.e., they cannot generate progeny virions but retain the ability to infect target cells.
• the genome of the viral vector does not include genes encoding the enzymes required to replicate (the genome can be engineered to be "gutless" - containing only the transgene of interest flanked by the signals required for amplification and packaging of the artificial genome), but these genes may be supplied during production.
• AAV GC number titration One method for performing AAV GC number titration is as follows: Purified AAV vector samples are first treated with DNase to eliminate un-encapsidated AAV genome DNA or contaminating plasmid DNA from the production process. The DNase resistant particles are then subjected to heat treatment to release the genome from the capsid. The released genomes are then quantitated by real-time PCR using primer/probe sets targeting specific region of the viral genome (usually poly A signal).
• DNA and RNA is generally measured in the nanogram (ng) to microgram
• IU per volume (e.g., mL).
• IU is infectious unit, or alternatively transduction units (TU);
• IU and TU can be used interchangeably as a quantitative measure of the titer of a viral vector particle preparation.
• the lentiviral vector is typically non-integrating.
• the amount of viral particles is at least about 3xl0 6 IU, and can be at least about lxl O 7 IU, at least about 3xl 0 7 IU, at least about 1x10 s IU, at least about 3x10 s IU, at least about lxl O 9 IU, or at least about 3x10 9 IU.
• the above-described recombinant vectors may be delivered to host cells according to published methods.
• the rAAV preferably suspended in a physiologically compatible carrier, may be administered to a human or non-human mammalian patient.
• suitable carriers may be readily selected by one of skill in the art in view of the indication for which the transfer virus is directed.
• one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline).
• Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present invention.
• compositions of the invention may contain, in addition to the rAAV and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers.
• suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.
• Suitable chemical stabilizers include gelatin and albumin.
• the viral vectors described herein may be used in preparing a medicament for delivering ornithine transcarbamylase to a subject (e.g., a human patient) in need thereof, supplying functional hOTCase to a subject, and/or for treating ornithine transcarbamylase deficiency.
• a course of treatment may optionally involve repeat administration of the same viral vector (e.g., an AAV8 vector) or a different viral vector (e.g., an AAV8 and an AAVrhlO). Still other combinations may be selected using the viral vectors and non-viral delivery systems described herein.
• RNA delivery technologies are also available, e.g., from Veritas Bio [see, e.g., US 2013/0323001, Dec 23, 2010, "In vivo delivery of double stranded RNA to a target cell" (cytosolic content including RNAs, e.g., mRNA, expressed siRNA/shRNA/miRNA, as well as injected/introduced siRNA/shRNA/miRNA, or possibly even transfected DNA present in the cytosol packaged within exovesicles and be transported to distal sites such as the liver)].
• RNAs e.g., mRNA, expressed siRNA/shRNA/miRNA, as well as injected/introduced siRNA/shRNA/miRNA, or possibly even transfected DNA present in the cytosol packaged within exovesicles and be transported to distal sites such as the liver
• Still other systems for in vivo delivery of RNA sequences have been described. See, e.g.,
• the invention provides an engineered hOTC mRNA of the mature sequence (at least about nt 99 - 1098) or the full-length of SEQ ID NO: 10, 11, 12, 13, or a sequence having at least 97% to 99% identity thereto, in a composition for delivery of double-stranded or single stranded RNA which results in expression of the mature hOTCase in a target host cell, e.g., a liver cell.
• a target host cell e.g., a liver cell.
• RNA live directly, as compared to transcribed from a DNA delivery molecule
• hOTCase live directly, as compared to transcribed from a DNA delivery molecule
• a cap or a modified base in order to avoid rapid clearance of the RNA, it is modified as described herein (e.g., using a cap or a modified base), such that its effects may be retained for over 24 hours, over 48 hours, or up to about 3 days (about 72 hours). It may be desirable to co-administer an mRNA directly as described herein and co-administer at the same or substantially the same time, a DNA or viral vector-based hOTC composition as defined herein.
• a subject may receive immediate treatment, and at such time as the mRNA-mediated expression begins to wane, the longer-term hOTC expression conferred by a viral vector - mediated expression begins to take effect.
• a subject may receive a second administration of an mR A-based composition as defined herein.
• the m NA compositions described herein may be used in other therapeutic regimens or methods, including those involving OTCD patients who are not in acute crisis.
• compositions according to the present invention may comprise a pharmaceutically acceptable carrier, such as defined above.
• the composition comprises a block copolymer associated with a hOTC polynucleotide as described herein.
• the block copolymers may form a micelle, such that the micelle comprises a plurality of block copolymer.
• such a composition contains a nucleic acid molecule comprising the mRNA sequence corresponding to the hOTC sequence encoding the mature hOTCase (at least about nt 99 to 1098) or the full-length of any of SEQ ID NO: 10, 11, 12, 13.
• this nucleic acid molecule may include the 5' untranslated region (UTR), also known as the leader sequence or leader RNA, and one or more of an optional intron(s), an optional exon(s), an optional a Kozak sequence, an optional WPRE. and a polyA, and the 3' UTR flanking the coding sequences.
• UTR 5' untranslated region
• Suitable leader sequences include those discussed above in connection with the hOTC DNA sequences, which discussion is incorporated by reference herein. Examples of sources of suitable leader sequences, other than the native hOTC leader sequences, or those corresponding to FIG 2, FIG3 or FIG4, or FIG 5 are discussed above. Similarly, sources of suitable introns, polyA, and Kozak sequences are discussed above and are applicable to the delivery of the corresponding RNA sequences discussed in the present paragraph. Further, various modifications to the RNA may be generated, e.g., a modified 5' cap structure may be engineered into the construct in order to avoid rapid clearance of the mRNA in vivo, or for another desired reason. Methods of generating such 5' cap structures is known to those of skill in the art.
• modified nucleotides can be used to make mRNA in vitro, like pseudouridine.
• RNA may be dosed repetitively, or subject can be dosed first with mRNA to manage neonatal crises followed up by viral vector-mediated delivery ⁇ e.g., AAV) for long term therapy and to prevent fibrosis/cirrhosis and/or hepatocellular carcinoma.
• viral vector-mediated delivery e.g., AAV
• RNAs are produced by SP6 in vitro transcription of cloned cDNAs, Nucleic Acids Res. 1984 Sep 25;12(18):7057-70; and Rio, D. C, et al. RNA: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 2011, 205-220. Each of these references is incorporated herein by reference. In addition, kits and protocols for generating mRNA are available
• RNA Synthesis Kit TriLink Biotechnologies; bioSYNTHESIS; GE Dharmacon; and IBA Lifesciences.
• translation in the context of the present invention relates to a process at the ribosome, wherein an mRNA strand controls the assembly of an amino acid sequence to generate a protein or a peptide.
• a "therapeutically effective amount" of the hOTC is delivered as described herein to achieve a desired result, i.e., treatment of OTC deficiency or one or more symptoms thereof.
• a desired result includes reducing orotic acid levels, reducing hyperammonemia and/or minimizing or eliminating one or more of the neurophysical complications including developmental delay, learning disabilities, intellectual disability, attention deficit hyperactivity disorder, and executive function deficits.
• Treatment may include treatment of subjects having severe neonatal-onset disease (males or, more rarely, females), and late-onset (partial) disease in males and females, which may present from infancy to later childhood, adolescence, or adulthood.
• an RNA based delivery system for functional hOTC is used to stabilize a subject (e.g., a human patient) in crisis, followed by delivery of a viral vector mediated delivery of a functional hOTC.
• initial therapy involves co-administration of viral and non-viral - mediated hOTC delivery systems.
• the hOTC DNA and RNA constructs may be used alone, or in combination with the standard of care for the patient's diagnosis and condition.
• treatment of the patient e.g., a first injection
• treatment is initiated prior to the first year of life.
• treatment is initiated after the first 1 year, or after the first 2 to 3 years of age, after 5 years of age, after 11 years of age, or at an older age.
• the method of the invention provides for treating and/or reversing liver fibrosis and/or OTCD-related cirrhosis by delivering to the subject a functional OTCase which is encoded by an engineered DNA of SEQ ID NO: 1, 3, 4, 5, 8 or 9, or a chimeric DNA as defined herein.
• Delivery of the DNA may be mediated by a viral vector containing the engineered in an expression cassette, or by a non-viral delivery system, either of which mediates expression of functional OTCase in the liver cells of the subject.
• the subject is administered an engineered RNA of SEQ ID NO: 10, 11, 12 or 13, or a chimeric RNA as defined herein. Delivery of the RNA may be mediated by a viral vector containing the engineered RNA in an expression cassette, or by a non-viral delivery system, either of which mediates expression of functional OTCase in the liver cells of the subject.
• the invention provides methods of preventing treating and/or preventing HCC by delivering to the subject (e.g., a human patient) a hOTC.
• This aspect of the invention may utilize a viral or non-viral delivery system.
• the nucleic acid expression cassette may contain a synthetic hOTC DNA or RNA as provided herein, or another suitable sequence which and expresses functional hOTCase.
• treatment of the patient e.g., a first injection
• treatment of the patient is initiated prior diagnosis with HCC.
• treatment of the patient is initiated following HCC diagnosis.
• treatment is involves co-administration with sorafenib (commercially available as Nexavar®), or being used in conjuction with chemoembolization, radiation, thermal ablation, precutanous ethanol injection, targeted therapy (e.g., anti-angiogenesis drugs), hepatic arterial infusion of anti-cancer drugs, immunotherapy, or with surgical options including, e.g., resection, cryosurgery, and liver transplant.
• OTCase such as characterized by SEQ ID NO: 2 or another OTCase which provides at least about 50%, at least about 75%, at least about 80%, at least about 90%, or about the same, or greater than 100% of the biological activity level of the wild-type human ornithine transcarbamylase enzyme, which may be characterized by the sequence of SEQ ID NO:2 or a natural variant or polymorph thereof which is not associated with disease. More particularly, as heterozygous patients may have as low an OTCase functional level as about 50% or lower, effective treatment may not require replacement of OTCase activity to levels within the range of "normal" or non-OTCD patients.
• regulation refers to the ability of a compound of formula (I) to inhibit one or more components of a biological pathway.
• pAAVsc.TBG.hOTCwt and pAAVsc.TBG.hOTCco-LW4 were constructed by replacing the mOTC coding sequencing with wild-type (WT) hOTC (hOTCwt) or hOTCcoLW cDNA, respectively, in a plasmid derived from the previously described pAAVsc.TBG.mOTC 1.3 with the intron disrupted [Moscioni D, et al, "Long- term correction of ammonia metabolism and prolonged survival in ornithine
• PEI polyethylenimine
• Genome titers [genome copies (GC)/ml] of AAV vectors were determined by real-time PCR using primer and probe sets targeting the TBG promoter (forward primer 5'-AAACTGCCAATTCCACTGCTG-3' [SEQ ID NO: 14], reverse primer 5 '-CCATAGGCAAAAGCACCAAGA-3 ' [SEQ ID NO:15 ], probe 6FAM- TTGGCCCAATAGTGAGAACTTTTTCCTGC [SEQ ID NO: 16] -TAMRA), and using a linearized plasmid as the standard.
• the forward primer is located 400bp downstream of the 5' closed hairpin.
• OTC protein expression levels and OTC activity were evaluated in the liver of spf ⁇ mice 14 days after i.v. injection of l x l O 11 GC of AAV2/8sc.TBG.hOTCwt or AAV2/8sc.TBG.hOTCco-LW4 vectors.
• the sp 811 mice are a model for late onset OTC disease in humans. All animal procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the IACUC.
• Enzyme activity was initiated with the addition of freshly prepared 50 mM carbamyl phosphate dissolved in 50 mM Tris-acetate pH 8.3, allowed to proceed for 5 minutes at 25 °C and quenched by addition of an equal volume of 5 mMl 3 C5 -citrulline in 30%TCA. Debris was separated by 5 minutes of microcentrifugation, and the supernatants were transferred to vials for mass spectroscopy.
• LW4 The vector carrying engineered hOTC cDNA termed herein LW4 (FIG. 4) was found to improve expressed hOTC protein levels by 100-fold.
• An assessment of OTC enzyme activity generally correlated with the OTC Western blot experiments although OTC protein was more elevated than OTC enzyme activity when compared to endogenous OTC.
• the hOTCco-LW4 resulted in over 33 -fold higher activity than the hOTCwt. Sustained and dose-correlated hOTC expression and activity levels were observed in the treated sp 11 mice.
• the illustrative vector carrying the modified hOTCco-LW4 (FIG. 4) provided high level of transduction, as measured by OTC histological assays, throughout a broad range of doses. Between doses l x lO 11 GC and 3x 10 9 GC, transduction efficiency, as measured by histochemical staining, varied between 50-70%. At the lowest dose of 1 x 10 9 GC, 40% of the liver areas were positive by OTC histochemical staining.
• pAAVsc.TBG.hOTCco is cloned into an AAV8 capsid [Gao et al, PNAS USA, 2002, 99:11854-11859] using conventional techniques.
• Plasmids containing the codon optimized hOTCco sequences are cloned as described by replacing the mOTC coding sequencing of the pLSPlmOTC plasmid [Cunningham et al, Mol Ther, 2009, 17: 1340-1346] with the corresponding cDNA sequence of SEQ ID NO:3, 4, 5, 9 or 10.
• the resulting plasmids pAAVsc.LSPl .hOTCco are cloned into AAV8 capsids to form the corresponding ssAAV2/8.LSPl .hOTC-co vectors using techniques described in Example 1.
• the vectors generated according to Part A or B may be purified by two rounds of cesium chloride gradient centrifugation, buffered-exchanged with PBS, and concentrated using Amicon Ultra 15 centrifugal filter devices- 100K (Millipore, Bedford, MA). Genome titer (GC/ml) of AAV vectors can be determined by real-time PCR using a primer/probe set corresponding to the TBG promoter and linearized plasmid standards.
• Vectors can be subject to additional quality control tests including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis for vector purity and Limulus amebocyte lysate (LAL) for endotoxin detection (Cambrex Bio Science, Walkersville, MD, USA).
• SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
• LAL Limulus amebocyte lysate
• the single-stranded vectors may have other desirable properties, e.g., in terms of kinetics, depending upon the age and condition of the patient.
• hOTCco cDNA [SEQ ID NO: 3, 4, 5, 9 and 10] with Notl linkers is cloned downstream of a rat PEPCK promoter to generate pPEPCK-hOTC as described in A. Mian et al, Molecular Therapy, 2004, 10: 492-499 (2004).
• This plasmid is digested with Ascl, and the resultant PEPCK-hOTCco fragment is inserted into the adenoviral backbone plasmid pC4HSU31 to generate the parental plasmids pC4HSU-PEPCK- hOTCco.
• Plasmid pWPRE is digested with Clal to release the WPRE, which is then inserted into the Mlul site of pPEPCK-hOTC, to generate pPEPCK-hOTCco-WPRE plasmid with their respective hOTCco sequences.
• the remaining steps to generate the adenoviral plasmid pC4HSU-PEPCK-hOTCco-WPRE are as previously described. All cloning sites are confirmed by DNA sequence analysis. The identity of recombinant adenoviral plasmids can be confirmed by restriction enzyme digestion with Hindlll and BamHI. The adenoviral plasmids are linearized with Pmel before transfection into 293Cre4 cells.
• Adenoviral vectors are rescued and amplified with 293Cre4 cells and helper virus AdLC8cluc. Suspension 293N3Scre8 cells may be used in the final step of vector production. Purification, quantification by OD260 and viral DNA extraction are performed as described in detail elsewhere [Brunetti-Pierri, N., et al. (2004). Acute toxicity after high-dose systemic injection of helper-dependent adenoviral vectors into nonhuman primates. Hum. Gene Ther. 15: 35 ⁇ 16; Ng, P., Parks, R. J. and Graham, F. L. (2002). Preparation of helper-dependent adenoviral vectors. Methods Mol. Med. 69: 371— 388].
• Replication-defective lentiviral vectors containing the hOTCco sequences provided herein can be produced by replacing the rat OTC gene sequence insert of the plasmid pLenti-GIII-CMV-GFP-2A-Puro [commercially available from Applied Biological Materials (ABM) Inc.; Canada] with the desired hOTCco sequence [SEQ ID NO: 3, 4, 5, 9 and 10].
• the viruses are generated according to manufacturer instructions.
• the ABM system includes an enhancer deletion in the U3 region of 3'ALTR to ensure self-inactivation of the lentiviral vector following transduction and integration into the target cell's genomic DNA; contains minimal lentiviral genes necessary for packaging, replication and transduction (Gag/Pol/Rev), derived from different plasmids all lacking packaging signals; further, none of the Gag, Pol, or Rev genes are incorporated into in the packaged viral genome, thus making the mature virus replication-incompetent.
• the hOTCco DNA of SEQ ID NO: 3, 4, 5 9 and 10 is cloned into a lentivirus pseudotyped with a vesicular stomatitis virus (VSV) envelope gene, purchased from InvivoGen (SanDiego, CA) using manufacturer's instructions.
• VSV vesicular stomatitis virus
• RNA may be prepared by in vitro transcription from a DNA template or synthesized.
• the RNA expression cassette is prepared which includes a 5 ' UTR, an optional intron with splice donor and acceptor sites, an optional Kozak sequence, the hOTC coding sequence provided herein, a polyA, and a 3' UTR using known techniques.
• a suitable amount of mRNA are incorporated into a lipid- enveloped pH-responsive polymer nanoparticles generated using published techniques. [X. Su et al, Mol. Pharmaceutics, 2011, 8 (3), pp 774-787; web publication: March 21, 2011].
• PEI polyethyleneinime
• a lipid formulation is created using expression cassette containing the hOTCco RNA in a formulation of cK - E12:DOPE:Chol:PEG-DMG2K (relative amounts 50:25:20:5 (mg:mg:mg:mg)) to provide a solution for delivery.
• the cationic lipid cK -E12 is used (see, e.g., WO 2013/063468), and is combined with dioleoylphosphatidyl-ethanolamine or "DOPE", cholesterol (chol), and polyethylene glycol (PEG) or a PEGylated lipid (PEG-DMG2K) using e formulation methods described in international patent publications WO 2010/053572 and WO 2012/170930, both of which are incorporated herein by reference. [000120]
• DOPE dioleoylphosphatidyl-ethanolamine
• chol cholesterol
• PEG polyethylene glycol
• PEG-DMG2K PEGylated lipid
• the hOTCco sequences [SEQ ID NO: 3, 4, or 5] are engineered as naked plasmid DNA constructs which are delivered to a target liver cell (e.g., via intravascular administration) and express the human OTC protein in the target cell.
• Cationic lipid-DNA complexes - Cationic lipid - DNA complexes are prepared using a suitable amount of an expression cassette containing at least a promoter, an optional intron, an optional Kozak sequences, an hOTCco of SEQ ID NO: 3, 4 or 5, a poly A, and other optional expression control sequences.
• the promoter may be a liver specific promoter. Alternatively, another non-tissue specific promoter may be selected.
• the scAAV8.TBG.hOTCcoLW4 prepared as described in Example 1 was used to rescue animals in a mouse model of neonatal (early) onset OTCD.
• OTC KO mice were generated through the deletion of exons 2-3, and the properties of this mouse characterized in terms of similarity to human patients with null mutations of OTC.
• the OTC knockout (KO) model generated in our laboratory through the deletion of exons 2-3 closely mimics the severe neonatal onset form of OTCD in humans.
• Neonatal male OTC KO pups have elevated plasma ammonia levels due to the absence of OTC expression in the liver, and they inevitably die within 24 hours after birth.
• a single injection of scAAV8-hOTCco vector prepared as described in Example 1 at a dose of l-3xl0el0 GC/pup immediately after birth is able to rescue the OTC KO pups and extend the life to 6 weeks.
• mice show close to normal levels of plasma ammonia, although urine orotic acid levels in a subset of these mice were significantly elevated.
• Sirius red staining on liver samples from heterozygous mice of different ages (6, 12, and 18 months old) showed liver fibrosis in aged (18-month old) OTC-KO heterozygous female mice, similar to a liver sample from a 11 -year-old OTCD patient.
• AAV8sc-hOTCco vector can prevent liver fibrosis in OTC-KO heterozygous and has great potential for correction of liver fibrosis in OTCD patients.
• Gene therapy vectors described herein are capable of rapid, robust and prolonged gene expression even in mice with a complete lack of OTC. Heterozygous females are able to reproduce and deliver hemizygous male offspring, but these pups die within a day of birth if untreated. Untreated old heterozygous female mice show evidence of increased fibrosis and microvesicular steatosis, a finding that appears similar to observations in human heterozygous patients. A regimen of gene transfer that is able to rescue affected males has been developed and treated males have survived over 72 weeks.
• liver-specific gene therapy with hOTC can prevent liver fibrosis.
• These data correlate with in treatment of heterozygous OTC deficient humans, e.g., subjects having late onset of OTCD.

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