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WO1999047650A2 - Polymerase d'arn purifiee d'enterobacter - Google Patents

Polymerase d'arn purifiee d'enterobacter Download PDF

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Publication number
WO1999047650A2
WO1999047650A2 PCT/US1999/006158 US9906158W WO9947650A2 WO 1999047650 A2 WO1999047650 A2 WO 1999047650A2 US 9906158 W US9906158 W US 9906158W WO 9947650 A2 WO9947650 A2 WO 9947650A2
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Prior art keywords
rna polymerase
enterobacter
purified
holoenzyme
cloacae
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PCT/US1999/006158
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WO1999047650A3 (fr
Inventor
Kelvin T. Lam
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Scriptgen Pharmaceuticals Inc
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Scriptgen Pharmaceuticals Inc
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Priority to AU33596/99A priority Critical patent/AU3359699A/en
Publication of WO1999047650A2 publication Critical patent/WO1999047650A2/fr
Publication of WO1999047650A3 publication Critical patent/WO1999047650A3/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1247—DNA-directed RNA polymerase (2.7.7.6)

Definitions

  • This invention relates to purified bacterial DNA-dependent RNA polymerases, particularly those from Enterobacter species, which are useful as targets for antibacterial drugs.
  • Enterobacteriaciae are Gram-negative rods that are the most common cause of urinary tract infections and are important etiological agents of diarrhea (Farmer III, 1995). Spread of Enterobacteriaciae to the bloodstream leads to Gram-negative sepsis.
  • the Enterobacteriaciae are a large, diverse family and include E. coli, Salmonella, Yersinia,
  • RNA polymerase holoenzyme consists of four subunits.
  • the core enzyme (containing two ⁇ subunits, a ⁇ subunit and a ⁇ ' subunit) binds to a ⁇ subunit to form a holoenzyme.
  • the primary ⁇ factor originally identified as a stimulating factor (Burgess, 1969), determines promoter specificity.
  • the core enzyme can initiate transcription only in a non-specific manner.
  • RNA polymerase has been purified from several bacterial species, including Escherichia coli (Burgess, 1969; Burgess & Jendrisak, 1975; Engbaek et al, 1976; Hager et al., 1990), Bacillus subtilis (Avila et al., 1971; Davison et al., 1979; Hager, et al., 1990), Pseudomonas aeruginosa (Allan & Kropinski, 1987), Pseudomonas putida (Johnson et al.,
  • RNA polymerase from Enterobacter there is a need in the art for purified RNA polymerase from Enterobacter Purified RNA polymerase can be used, e g , to identify antibacterial agents for prevention and treatment of enterobacterial infections
  • the present invention provides purified RNA polymerase from Enterobacter species, preferably E. cloacae
  • the invention encompasses purified RNA polymerase core enzyme (i e , containing ⁇ , ⁇ , and ⁇ ' subunits), purified holoenzyme (i e , containing, ⁇ , ⁇ , and ⁇ 'subunits, and ⁇ factor), and individually purified subunits
  • the invention also provides recombinant ⁇ , ⁇ , and ⁇ ' subunits and ⁇ factor, all of which may further comp ⁇ se sequences useful as purification tags, such as, e g , polyhistidine sequences
  • the invention encompasses methods for purification of Enterobacter RNA polymerase holoenzyme, core enzyme, and subunits In one embodiment, the method is carried out using the steps of
  • the invention encompasses screening methods to identify candidate antibacterial agents.
  • the method comprises
  • RNA polymerase subunits comprising one or more purified RNA polymerase subunits, core enzyme, or holoenzyme and components necessary for in vitro transcription
  • the method comprises (i) contacting purified RNA polymerase core enzyme, holoenzyme, or one or more individual subunits with a plurality of test compounds, and
  • Figure 1 A is a graphic illustration of the elution profile of E. cloacae RNA polymerase from a Bio-Gel Al 5 column Fifty ⁇ l aliquots of each fraction were used to assay promoter-specific transcriptional activity (solid line) The absorbance at 280 nm was measured
  • Figure IB is a graphic illustration of the elution profile of E. cloacae RNA polymerase from a Heparin agarose column Elution was carried out using a linear gradient of 0 1- 0 6 M KC1, starting at fraction #24 Fifty ⁇ l aliquots were used to assay promoter- specific transcription (solid line with squares) and non-specific transcription (solid line with open circles) The absorbance at 280 nm was measured for every other fraction (dotted line)
  • FIG. 2 is a photographic illustration of SDS-PAGE analysis of fractions obtained during purification of the E. cloacae RNA polymerase The proteins were separated on a 4-15% gradient acrylamide gel Lane 1, molecular weight markers, lane 2, ammonium sulfate precipitate of the supernatants of soluble fractions, lane 3, Bio-Gel A 1 5 pooled fractions, lane 4, purified E. cloacae RNA polymerase (RNAP), and lane 5, purified E. coli RNA polymerase
  • Figure 3 A is a graphic illustration of the effect of KC1 (rnM) on enzymatic properties of purified E. cloacae RNA polymerase Both promoter-specific and non-specific in vitro transcription activity were measured (using pTac and CT, respectively, as indicated)
  • Figure 3B is a graphic illustration of the effect of MgCl 2 (mM) on enzymatic activity
  • Figure 3C is a graphic illustration of the effect of DTT (mM) on enzymatic activity
  • Figure 3D is a graphic illustration of the effect of temperature on enzymatic activity
  • Figure 4 is a graphic illustration of the broad-spectrum activity of rifamycin against purified RNA polymerases derived from Eschenchia coli (solid squares), Pseudomonas aeruginosa (solid circles), Enterobacter cloacae (solid triangles), Staphylococus aureus (open squares), Enterococcus faecium (open circles), and Bacillus subtilhs (open triangles)
  • Figure 5 is a photographic illustration of an immunoblot of RNA polymerases purified from different source organisms The blots were stained using polyclonal antibodies against E. coli ⁇ 70 factor Lane 1, prestained molecular weight markers, lane 2, Escherichia coli, lane 3, Enterobacter cloacae, lane 4, Pseudomonas aeruginosa, lane 5, Staphylococcus aureus, lane 6, Bacillus subtilhs, lane 7, Enterococcus faecium
  • Figures 6A and 6B are a graphic illustration of RNA polymerase activity of E. cloacae core polymerase reconstituted in vitro with E. coli ⁇ factor, in the presence (A) or absence (B) of tac promotor RNA polymerase activity was expressed as the counts per minute of [ ⁇ - 32 P] UTP incorporated after 30 minutes of polymerase activity at 37 °C
  • Figure 7A is a photographic illustration of promoter-specific transc ⁇ ption products displayed on PAGE Lane 1, in vitro transcription using purified E. cob RNA polymerase, lane 2, in vitro transcription using purified E. cloacae RNA polymerase
  • Figure 7B shows the DNA template used in the reaction
  • the present invention advantageously provides purified Enterobacter DNA-dependent RNA polymerase, and methods for purifying the core enzyme and the holoenzyme
  • the purified RNA polymerase (RNAP) is useful for screening for inhibitors, which may provide anti-bacterial drugs that avoid or overcome antibiotic resistance problems
  • RNA polymerase subunit that is "derived from" a particular organism is a polypeptide encoded by the genome of that organism
  • RNA polymerase holoenzyme contains, in addition to the ⁇ , ⁇ , and ⁇ ' subunits, a ⁇ factor polypeptide
  • the invention encompasses purified core enzyme, purified holoenzyme, and individually purified subunits RNA polymerase holoenzyme according to the invention may comprise subunits derived from different species, such as, e g , a holoenzyme having a ⁇ subunit
  • Enterobacter RNA polymerase may be purified from wild-type or mutant Enterobacter cells, or from heterologous organisms or cells (including, but not limited to, bacteria, fungi, insect, plant, and mammalian cells) into which an
  • RNA polymerase-encoding sequence has been introduced and expressed Furthermore, the polypeptides may be part of recombinant fusion proteins
  • RNAP genes are well known in the art, e.g., as described above (see, e.g., Sambrook et al., 1989, supra)
  • the DNA may be obtained by standard procedures known in the art from cloned DNA (e.g., a DNA "library”), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired bacterium (See, for example, Sambrook et al., 1989, supra, Glover, D M (ed ), 1985, DNA Cloning A Practical
  • the gene or genes should be molecularly cloned into a suitable vector for propagation of the gene
  • Identification of the specific DNA fragment containing the desired Enterobacter RNAP gene may be accomplished in a number of ways. For example, a portion of an Enterobacter RNAP gene can be purified and labeled to prepare a labeled probe, and the generated DNA may be screened by nucleic acid hybridization to the labeled probe (Benton and Davis, Science 196: 180, 1977; Grunstein and Hogness, Proc. Natl. Acad. Sci. U.S.A. 72:3961, 1975). Alternatively, degenerate probes derived from the Enterobacter RNAP amino acid sequences can be prepared and used for hybridization to possible cloned sequences. Those DNA fragments with substantial homology to the probe will hybridize.
  • expression cloning methods can be used to identify the Enterobacter RNAP coding sequence, including detecting expression from an expression library using an -Enterobacter RNAP antibodies (described infra).
  • selection can be carried out on the basis of the properties of the gene, e.g., if the gene encodes a protein product having the isoelectric, electrophoretic, amino acid composition, partial or complete amino acid sequence, antibody binding activity, or ligand binding profile of Enterobacter RNAP protein as disclosed herein.
  • the presence of the gene may be detected by assays based on the physical, chemical, immunological, or functional properties of its expressed product.
  • the identified and isolated gene can then be inserted into an appropriate cloning vector.
  • vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Examples of vectors include, but are not limited to, E. coli, bacteriophages such as lambda derivatives, or plasmids such as pBR322 derivatives or pUC plasmid derivatives, e.g., pGEX vectors, pmal-c, pFLAG, etc.
  • the insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini.
  • the ends of the DNA molecules may be enzymatically modified.
  • any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences.
  • Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated.
  • the cloned gene is contained on a shuttle vector plasmid, which provides for expansion in a cloning cell, e.g., E. coli, and facile purification for subsequent insertion into an appropriate expression cell line, if such is desired.
  • a shuttle vector which is a vector that can replicate in more than one type of organism, can be prepared for replication in both E. co and Saccharomyces cerevisiae by linking sequences from an E. coli plasmid with sequences form the yeast 2m plasmid
  • Enterobacter RNA polymerase holoenzyme is purified from a bacterial culture by the following steps
  • RNA polymerase-containing fractions are adsorbed to, and specifically eluted from, a heparin column (See, e g , Example 1 below)
  • the invention also provides recombinant ⁇ , ⁇ , and ⁇ ' subunits and ⁇ factor, all of which may further comprise sequences useful as purification tags, such as, e g , polyhistidine sequences Purification of histidine-tagged subunits is achieved using affinity chromatography on, e g , Ni +2 -NTA-agarose (Qiagen) Purified RNA polymerase holoenzyme, core enzyme, or subunits may be modified in any manner known in the art, such as, for example, phosphorylation, sulfation, acylation, or other protein modifications They may also be modified with a label capable of providing a detectable signal, either directly or indirectly, including, but not limited to, radioisotopes and fluorescent compounds
  • Antibodies to Enterobacter RNAP are useful, / «ter aha, for diagnostics, detecting expression of Enterobacter RNAP (e.g., for cloning), and for screening
  • Enterobacter RNAP polypeptides may be used as an lmmunogen to generate antibodies that recognize the Enterobacter RNAP or a polypeptide thereof
  • Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library
  • RNAP polypeptides Various procedures known in the art may be used for the production of polyclonal antibodies to Enterobacter RNAP polypeptides.
  • various host animals can be immunized by injection with the Enterobacter RNAP polypeptide, or a derivative (e.g., fragment or fusion protein) thereof, including but not limited to rabbits, mice, rats, sheep, goats, etc.
  • the Enterobacter RNAP polypeptide or fragment thereof can be conjugated to an immunogenic carrier, e.g., bovine serum albumin
  • BSA keyhole limpet hemocyanin
  • KLH keyhole limpet hemocyanin
  • Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG
  • any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used (Kohler and Milstein, Nature 256:495-497, 1975; Kozbor et al.. Immunology Today 4:72, 1983; Cote et al, Proc. Natl. Acad. Sci. U.S.A.
  • Antibody fragments which contain the idiotype of the antibody molecule can be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab 1 fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • screening for or testing with the desired antibody can be accomplished by techniques known in the art, e.g., radioimmunoassay,
  • antibody binding can be detected by detecting a label on the primary antibody.
  • the primary antibody can be detected by detecting binding of a secondary antibody or reagent to the primary antibody
  • the secondary antibody is labeled
  • Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention
  • the foregoing antibodies can be used in methods known in the art relating to the localization and activity of the Enterobacter RNAP polypeptide, e.g., for Western blotting, imaging Enterobacter RNAP polypeptide in situ, measuring levels thereof in appropriate physiological samples, etc using any of the detection techniques mentioned above or known in the art
  • Such antibodies can also be used in assays for ligand binding, e.g., as described in US Patent No 5,585,277 and Patent No 5,679,582
  • such antibodies can be used to affinity purify Enterobacter RNAP or components thereof
  • nucleotide sequences derived from the gene encoding a polymorphic form o ⁇ a Enterobacter RNAP, and Enterobacter RNAP protein are useful targets to identify drugs that are effective in treating Enterobacter infections, particularly infection with antibiotic resistance bacteria
  • Drug targets include without limitation (i) isolated nucleic acids derived from the gene encoding a Enterobacter RNAP, (ii) isolated derived from Enterobacter RNAP, and (iii) Enterobacter RNAP (core enzyme or holoenzyme)
  • Purified Enterobacter RNA polymerase holoenzyme, core enzyme, or individual subunits according to the invention can be used in screening methods to identify candidate antibacterial agents
  • in vitro transcription is carried out using one or more purified Enterobacter RNA polymerase subunits, core enzyme, or holoenzyme, in a mixture containing components necessary for in vitro transcription
  • a plurality of test compounds are added to the mixture prior to initiation of the reaction, and the effect of the compounds on transcription is monitored.
  • Candidate antibacterial compounds are those compounds that modify, preferably decrease, the transcriptional activity of the mixture.
  • the screening method comprises contacting purified RNA polymerase core enzyme, holoenzyme, or one or more individual subunits with a plurality of test compounds; and measuring the binding of the compounds to the RNA polymerase or subunit.
  • Candidate antibacterial compounds are those compounds that bind to the RNA polymerase or subunits thereof.
  • the screening methods of the present invention are adapted to a high-throughput format, allowing a multiplicity of compounds to be tested in a single assay.
  • inhibitory compounds may be found in, for example, natural product libraries, fermentation libraries (encompassing plants and microorganisms), combinatorial libraries, compound files, and synthetic compound libraries.
  • synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford, CT).
  • a rare chemical library is available from Aldrich Chemical Company, Inc.
  • RNA polymerase inhibitors using the methods of the present invention may be modified to enhance potency, efficacy, uptake, stability, and suitability for use in pharmaceutical formulations, etc. These modifications are achieved and tested using methods well-known in the art.
  • RNA Polymerase Holoenzyme and Core Enzyme From Enterobacter Cloacae Enterobacter cloacae RNA polymerase was purified as follows
  • a culture ofE. cloacae strain ATCC 13047 (obtained from the American Type Culture Collection) was grown to early log phase (A ⁇ - 0 4-0.6) in LB medium and the cells were harvested by centrifugation
  • the bacterial paste was weighed (100 g) and lysed (approximately 1 1 weight/volume) using a French Press (10,000-15,000 p s i) in 0.2 M KCl in TG ⁇ D (50 mM Tris-HCl, pH 7 5, 10% glycerol, 1 mM ⁇ DTA 10 mM DTT and 10 mM MgCl 2 )
  • the crude extract was centrifuged at 10,000 x g for 30 min at 4°C
  • the supernatant solution was collected and 3 5 ml of 10% polyethylenimine (P ⁇ I) (Sigma Chemical Co ) was added per 100 ml of supernatant
  • P ⁇ I polyethylenimine
  • the solution was stirred gently for 30 min at 4°C and centrifuged at 10,000 x g for 30 min at 4°C
  • the pellet was resuspended with 30 ml of 0 5 M KCl in TG ⁇ D and centrifuged at 10,000 x g for 30 min at 4°C
  • the pellet was resuspended with TG ⁇ D containing 1 M KCl and centrifuged at 10,000 x g for 30 min at 4°C
  • the supernatant was collected and 35 g of ammonium sulfate was added per
  • transcription reactions were incubated at 37°C for 30 min, after which newly transcribed RNA was precipitated onto glass fiber filter membrane using TCA (trichloroacetic acid) and radioactivity determined by scintillation counting
  • Figure 1A shows the size separation of RNA polymerase activity on the Bio- Gel column. A single peak of promoter-specific activity was observed
  • cloacae RNA polymerase (lane 4) contains four polypeptides having molecular masses of approximately 156, 151, 82 and 45 kDa Without wishing to be bound by theory, based on their similarity in size to the corresponding E. coh. subunits, these polypeptides are identified as the ⁇ ', ⁇ , ⁇ , and ⁇ subunits, respectively, of E. cloacae RNA polymerase
  • holoenzyme was dialyzed against TGED containing 0 1 M KCl
  • the sample was loaded onto a 1 5 ml Bio-Rex70 column (BioRad, Hercules CA) pre-equilibrated with TGED containing 0 1 M KCl
  • the column was washed with TGED containing 0 1 M KCl, and the core polymerase was eluted with 0 6M KCl TGED buffer
  • the flow through contained the ⁇ factor and the eluted peak contained core enzyme, as determined by SDS-PAGE analysis
  • the enzymes were concentrated and dialyzed against TGED containing 50% glycerol and 0 5M KCl This procedure efficiently separated the ⁇ factor from the core enzyme
  • E. cloacae RNA polymerase holoenzyme purified as described in Example 1 above was characterized as follows 1 Reaction conditions The optimum KCl. MgCl ? , and DTT concentrations, as well as the optimum temperature for E.
  • RNA polymerase activity were determined using in vitro transcription reactions containing pTac DNA as a template
  • the KCl concentration was varied from 10 mM to 400 mM in the presence of 10 mM MgCl 2 and 10 mM DTT
  • the optimum KCl concentration under these conditions was between 200 and 300mM ( Figure 3 A)
  • the optimum MgCl 2 concentration was determined to be between 5 and 15 mM ( Figure 3B)
  • RNA polymerase retained full activity in buffers containing up to 100 mM DTT ( Figure 3C)
  • the optimum temperature was determined to be 40°C ( Figure 3D)
  • ⁇ 70 expression was induced by the addition of isopropylthio- ⁇ -D-galactoside (LPTG) to a concentration of 2 mM, after which the culture was shaken for an additional 3 hr at 37° C.
  • the culture was harvested by centrifugation (3000 x g; 30 min at 4°C), and the cell pellet was resuspended in 20 ml buffer A (20 mM Tris- HCl (pH 8.0), 500 mM NaCl, 5 mM imidazole).
  • the cells were lysed by sonication, and the lysate was cleared by centrifugation (16,000 x g; 30 min at 4°C).
  • N-terminally histidine- tagged ⁇ 70 was precipitated by addition of ammonium sulfate to 60% saturation and collected by centrifugation (16,000 x g; 20 min at 4°C). The pellet was redissolved in buffer A containing 6M guanidine hydrochloride. The sample was adsorbed onto 1 mL of Ni 2+ -NTA agarose (Qiagen, Chatsworth, CA) equilibrated with buffer A, washed three times with buffer A followed by three washes with buffer B (50 mM Tris-HCl (pH 8), 200 mM KCl, 10 mM
  • the anti-E. coli ⁇ 70 antibodies prepared as described above were used to detect cross-reactivity between E. cloacae and E. coli RNA polymerases using immunoblots. Proteins were resolved by SDS-PAG ⁇ analysis and transferred to nitrocellulose membrane
  • Membranes were treated with 1% BSA in Tris-buffered saline containing 0.1% Tween 20 (TBST) for one hour. Membranes were incubated with antibodies diluted with TBST for 2 hours. After washing, the membranes were treated with goat anti-rabbit alkaline phosphatase-conjugated antibody (Amersham, Arlington Heights, IL) for 60 minutes. After washing, antigen-antibody complexes were detected by soaking the membrane in NBT/BCLP solution for 10 mininutes.
  • TST Tris-buffered saline containing 0.1% Tween 20

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Abstract

La présente invention concerne une polymérase purifiée d'ARN dépendant de l'ADN d'Enterobacter. L'invention concerne plus particulièrement une haloenzyme et une enzyme noyau de la polymérase d'ARN provenant d'Enterobacter cloacae. L'invention concerne également un procédé permettant de faire une recherche systématique d'agents antibactériens se liant à une polymérase de l'ARN dépendant de l'ADN d'Enterobacter.
PCT/US1999/006158 1998-03-16 1999-03-16 Polymerase d'arn purifiee d'enterobacter Ceased WO1999047650A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU33596/99A AU3359699A (en) 1998-03-16 1999-03-16 Purified rna polymerase from (enterobacter)

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US7820398P 1998-03-16 1998-03-16
US60/078,203 1998-03-16

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WO1999047650A2 true WO1999047650A2 (fr) 1999-09-23
WO1999047650A3 WO1999047650A3 (fr) 2000-01-20

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11926817B2 (en) 2019-08-09 2024-03-12 Nutcracker Therapeutics, Inc. Microfluidic apparatus and methods of use thereof

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DE3401619A1 (de) * 1984-01-18 1985-07-18 Boehringer Mannheim Gmbh, 6800 Mannheim Restriktionsendonuklease mae ii, ihre gewinnung und verwendung
US5583026A (en) * 1994-08-31 1996-12-10 Cornell Research Foundation, Inc. Process for reconstituting the polymerase III* and other subassemblies of E. coli DNA polymerase III holoenzyme from peptide subunits
AU4759596A (en) * 1995-02-15 1996-09-04 Research Foundation Of The State University Of New York, The Method for treatment of disease with anti-sigma factor asia

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11926817B2 (en) 2019-08-09 2024-03-12 Nutcracker Therapeutics, Inc. Microfluidic apparatus and methods of use thereof
US12448618B2 (en) 2019-08-09 2025-10-21 Nutcracker Therapeutics, Inc. Microfluidic apparatus and methods of use thereof

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AU3359699A (en) 1999-10-11

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