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  Anti-infective therapyUSPTO Application #: 20080026426Title: Anti-infective therapy Abstract: DNA isolates coding for human DNase and methods of obtaining such DNA are provided, together with expression systems for recombinant production of human DNase useful in therapeutic or diagnostic compositions. (end of abstract) Agent: Genentech, Inc. - South San Francisco, CA, US Inventor: Steven Shak USPTO Applicaton #: 20080026426 - Class: 435069100 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Micro-organism, Tissue Cell Culture Or Enzyme Using Process To Synthesize A Desired Chemical Compound Or Composition, Recombinant Dna Technique Included In Method Of Making A Protein Or Polypeptide The Patent Description & Claims data below is from USPTO Patent Application 20080026426. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This invention is a continuation-in-part of U.S. Ser. No. 07/289958 filed 23 Dec. 1988. This invention relates to new methods for making deoxyribonuclease (DNase), especially human DNase, and to nucleic acid encoding DNase. [0002] DNase is a phosphodiesterase capable of hydrolyzing polydeoxyribonucleic acid. It acts to extensively and non-specifically degrade DNA and in this regard is distinguished from the relatively limited and sequence-specific restriction endonucleases. This invention is concerned principally with DNase I and II. DNase I has a pH optimum near neutrality, an obligatory requirement for divalent cations, and produces 5'-phosphate nucleotides on hydrolysis of DNA. DNase II exhibits an acid pH optimum, can be activated by divalent cations and produces 3'-phosphate nucleotides on hydrolysis of DNA. Multiple molecular forms of DNase I and II also are known. [0003] DNase from various species has been purified to varying degree. Bovine DNase A, B, C, and D was purified and completely sequenced as early as 1973 (Liao et al., J. Biol. Chem. 248:1489 [1973]; Salnikow et al., J. Biol. Chem. 248:1499 [1973]; Liao et al., J. Biol. Chem. 249:2354 [1973]). Porcine and ovine DNase have been purified and fully sequenced (Paudel et al., J. Biol. Chem. 261:16006 [1986] and Paudel et al., J. Biol. Chem. 261:16012 [1986]). Human urinary DNase was reported to have been purified to electrophoretically homogeneous state and the N-terminal amino acid observed to be leucine; no other sequence was reported (Ito et al., J. Biochem. 95:1399 [1984]; see also Funakoshi et al., J. Biochem. 82:1771 [1977]; Murai et al., Biochim. et Biophys. Acta 517:186 [1978] and Wroblewski et al., P.S.E.B.M. 74:443 [1950]). [0004] Notwithstanding that full sequence information for a mammalian DNase first became known in 1973, only recently has a report appeared of an attempt to clone and express this class of enzymes. Shields et al. describe the expression cloning of part of the gene for bovine DNase I and expression of a fusion product in E. coli which was biologically and immunologically active (Biochem. Soc. Trans. 16:195 [1988]). The DNase product of Shields et al., however, was toxic to the host cells and could only be obtained by the use of an inducible promoter. Furthermore, great difficulty was encountered in attempts to isolate plasmid DNA from either clone, an obstacle attributed to constitutive levels of expression of DNase from the clones, so that these authors were unable to determine the sequence for the DNase-encoding nucleic acid. According to Shields et al., the inability to recover the plasmid was the result of constitutive expression of DNase even when the promoter was repressed at low temperature. This would create a considerable obstacle since Shields et al. had only identified the clone by expression cloning, which necessarily requires that the DNase be placed under the control of a promoter of some sort. [0005] DNase finds a number of known utilities, and has been used for therapeutic purposes. Its principal therapeutic use has been to reduce the viscosity of pulmonary secretions in such diseases as pneumonia, thereby aiding in the clearing of respiratory airways. Obstruction of airways by secretions can cause respiratory distress, and in some cases, can lead to respiratory failure and death. Bovine pancreatic DNase has been sold under the tradename Dornavac (Merck), but this product was withdrawn from the market. Reports indicate that this product had some clinical efficacy. However, although some clinicians observed no significant side effects (Lieberman, JAMA 205:312 [1968]), others noted serious complications such as pulmonary irritation and anaphylaxis (Raskin, Am. Rev. Resp. Dis., 98:697 [1968]). Such complications may be attributed to the fact that the previously marketed products were contaminated with proteases and were immunogenic in humans. In fact, although the clinical problem of thick pulmonary secretions is often chronic and recurring, prolonged therapy with bovine pancreatic DNase was not recommended. These problems could be overcome by providing DNase of human origin and producing it in large quantities in nonpancreatic exocrine cells to facilitate purification free of contaminant proteases. [0006] Accordingly, it is an object of this invention to provide nucleic acid encoding human DNase. [0007] It is another object to provide a method for expression of human DNase in recombinant cell culture. [0008] A further object is to enable the preparation of DNase having variant amino acid sequences or glycosylation not otherwise found in nature, as well as other derivatives of DNase, having improved properties including enhanced specific activity. SUMMARY OF THE INVENTION [0009] The objects of this invention have been accomplished by a method comprising providing nucleic acid encoding human DNase; transforming a host cell with the nucleic acid; culturing the host cell to allow DNase to accumulate in the culture; and recovering DNase from the culture. Surprisingly, a full length clone encoding human DNase has been identified and recovered, and moreover this DNA is readily expressed by recombinant host cells. [0010] In preferred embodiments the mammalian DNase is full-length, mature-human DNase, having the amino acid sequence of native human DNase, its naturally occurring alleles, or predetermined amino acid sequence or glycosylation variants thereof. The nucleic acid encoding the DNase preferably encodes a preprotein which is processed and secreted from host cells, particularly mammalian cells. BRIEF DESCRIPTION OF THE FIGURES [0011] FIG. 1 depicts the amino acid and DNA sequence of human DNase. The native signal sequence is underlined, the potential initiation codons are circled, and the mature sequence is bracketed. [0012] FIG. 2 shows a comparison between the amino acid sequence for mature human (hDNase) and bovine (bDNase) DNase. Asterisks denominate exact homology, periods designate conserved substitutions. [0013] FIG. 3 shows the construction of the expression vectors pRK.DNase.3 and pSVe.DNase. [0014] FIG. 4 shows the construction of the expression vector pSVI.DNase that contains the splice unit of the pRK5 vector without any modifications. [0015] FIG. 5 shows the construction of the expression vectors pSVI12.DNase, pSVI3.DNase, pSVI5.DNase, and pSV16b.DNase containing the modifications in the splice unit and surrounding DNA. [0016] FIG. 6 shows the complete nucleotide sequence of pSVI.DNase up to, but not including, the coding region of DNase. [0017] FIG. 7 shows the complete nucleotide sequence of pSVI2.DNase up to, but not including, the coding region of DNase. [0018] FIG. 8 shows the complete nucleotide sequence of pSVI3.DNase up to, but not including, the coding region of DNase. [0019] FIG. 9 shows the complete nucleotide sequence of pSVI5.DNase up to, but not including, the coding region of DNase. [0020] FIG. 10 shows the complete nucleotide sequence of pSVI6B.DNase up to, but not including, the coding region of DNase. [0021] FIG. 11 shows a schematic representation of the splice unit nucleotide sequences involved in the preparation of the vectors of this example, i.e., SVI, SVI2, SVI3, SVI5, and SVI6B. The boxes represent changes from the SVI sequence, the double underlining is a spurious ATG codon, the underlining shows spurious splice sites and added or changed branchpoint sequence (BPS) regions, the breaks in sequence represent deletions of the nucleotides for SVI3-SVI5, the " . . . " designation indicates sequence not shown, and the carets indicate the 5' and 3' cleavage sites within the splice donor and splice acceptor, respectively, of the splice unit. DETAILED DESCRIPTION Continue reading... Full patent description for Anti-infective therapy Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Anti-infective therapy patent application. Patent Applications in related categories: 20080166755 - Intracellular delivery of osteoinductive proteins and peptides - The invention provides fusion polypeptides comprising protein transduction domains and osteoinductive polypeptides, as well as methods of using such polypeptides to induce osteogenesis and to promote proteoglycan synthesis. The invention also provides osteoinductive peptides which have demonstrated the ability to induce bone formation in vivo. ... ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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