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Anti-infective therapy

a technology of anti-infective therapy and anti-tumor, which is applied in the field of anti-infective therapy, can solve the problems of not being able to determine the sequence of dnase-encoding nucleic acids, unable to obtain dnase products from shields et al., and being toxic to host cells, etc., and achieves the effect of being easily expressed

Inactive Publication Date: 2008-01-31
GENENTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention is a method for producing human DNase using a nucleic acid encoding the enzyme. The method involves transforming a host cell with the nucleic acid, culturing the host cell to allow DNase to accumulate in the culture, and then recovering the DNase from the culture. The recovered DNase is a full length clone that is readily expressed by recombinant host cells. The invention provides a way to produce a full-length, mature-human DNase that is suitable for use in various applications.

Problems solved by technology

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.
Obstruction of airways by secretions can cause respiratory distress, and in some cases, can lead to respiratory failure and death.
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.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Cloning of Human Pancreatic DNase I

cDNA Library Preparation

[0093] A human pancreatic cDNA library was constructed in λgt10 using polyadenylated mRNA prepared from freshly obtained and liquid N2 frozen human pancreas (Lauffer et al., Nature 318:334 [1985]). Using oligo dT primers and EcoRI-SalI-XhoI-SstII adapters, a cDNA library of 0.9×106 independent isolates of greater than 600 bp was obtained.

Oligonucleotide Probes

[0094] Two long probes were synthesized based on the amino acid sequence of bovine DNase I. Two segments of amino acid sequence which exhibited low redundancy were selected and mammalian codon usage tables were employed.

Probe 1:5′GTG-CTG-GAC-ACC-TAC-CAG-TAT-GAT-GAT-GGC-TGT-GAG-TCC-TGT-GGC-AAT-GAC 3′ (51 mer correspondingto the amino sequenceVal-Leu-Asp-Thr-Tyr-Gln-Tyr-Asp-Asp-Gly-Cys-Glu-Ser-Cys-Gly-Asn-Asp)Probe 2:5′TAT-GAC-GTC-TAC-CTG-GAC-GTG-CAG-CAG-AAG-TGG-CAT-CTG-AAT-GAT-GTG-ATG-CTG-ATG-GGC-GAC-TTC-AAC-GC3′ (71 mer corresponding to the amino acidsequence Ty...

example 2

Assays for DNase Activity

[0102] In addition to standard ELISA (see example 4) and RIA, three assays have been developed to detect DNase activity.

[0103] 1. Hydrolysis of 32P-labeled DNA. Radiolabeled 32P-DNA was prepared using an M13 single-stranded template, a 17mer sequencing primer, 32P-dCTP, non-radioactive DATP, dTTP, and dGTP, and Klenow. Briefly, 1.5λ MgCl2 (35 mM), 1.5λ 10× restriction buffer (70 mM Tris-HCl, pH 7.6; 35 mM dithiothreitol; 1 mM EDTA), and 6λ H2O were mixed with 1 template (approximately 1 μg) and 1.5λ Palmer (0.5 μM) and heated to 55° C. for 7 min. Nucleotide mix was prepared by taking 40λ32P-dCTP (sp. act.3000 Ci / mmol; 400 μCi) plus 1λ each of 2 mM stocks of non-radioactive DATP, dTTP, and dGTP to dryness and then reconstituting the nucleotides in 7λ 1× restriction buffer. The nucleotide mix and 1λ Klenow were added to the template-primer mixture and the reaction was incubated at 37° C. After 15 min, 2λ of a non-radioactive deoxynucleotides were added and t...

example 3

Expression of Human DNase I

1. pRK.DNase.7

[0108] Plasmid pRK.DNase.7 was constructed from clone 18-1, described above, as follows:

[0109] The plasmid pRK5 was digested with EcoRI, dephosphorylated, and fragment 1 comprising the bulk of the plasmid was isolated. pRK5 is described in Suva et al., Science 237:896 (1987); U.S. Ser. No. 97,472, filed Sep. 11, 1987; and EP Publ. 307,247, published 15 Mar. 1989, where the pCIS2.8c28D starting plasmid is described in EP 278,776 published Aug. 17, 1988 based on U.S. Ser. Nos. 07 / 071,674 and 06 / 907,297. The λ DNase clone 18-1 was digested with EcoRI and the insert (fragment 2) was isolated. Fragment 1 and fragment 2 were ligated and the ligation mixture transformed into E. coli strain 294. The transformed culture was plated on ampicillin media plates and resistant colonies selected. Plasmid DNA was prepared from transformants and checked by restriction analysis for the presence of the correct fragment.

[0110] pRK.DNase.7 was transfected int...

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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.

Description

[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 puri...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P21/00C07H21/04C12N15/63C12N5/10C12N15/09A61K38/00A61K38/21A61K38/46A61K45/06A61L29/00A61P11/00C12N1/15C12N1/19C12N1/21C12N9/16C12N9/22C12N11/08C12N15/55C12N15/71C12N15/83C12N15/85C12Q1/68C12R1/91
CPCA61K38/00C12N9/22C12N11/08C12N15/71C12Q1/6876C12N2800/108C12N2830/42C12N2840/44C12N15/85A61P11/00
Inventor SHAK, STEVEN
Owner GENENTECH INC
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