WO2002034892A1 - Bacteriophage modifie comprenant une modification non lytique et exprimant un gene kil - Google Patents

Bacteriophage modifie comprenant une modification non lytique et exprimant un gene kil Download PDF

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Publication number
WO2002034892A1
WO2002034892A1 PCT/EP2001/012169 EP0112169W WO0234892A1 WO 2002034892 A1 WO2002034892 A1 WO 2002034892A1 EP 0112169 W EP0112169 W EP 0112169W WO 0234892 A1 WO0234892 A1 WO 0234892A1
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phage
gene
modification
kil
dna
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PCT/EP2001/012169
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English (en)
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Udo BLÄSI
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Intercell Biomedizinische Forschungs- Und Entwicklungs Ag
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Publication of WO2002034892A1 publication Critical patent/WO2002034892A1/fr

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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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/00061—Methods of inactivation or attenuation
    • C12N2770/00062—Methods of inactivation or attenuation by genetic engineering

Definitions

  • the present invention relates to a modified phage, a modified phage DNA as well as a method for producing the modified phage.
  • Bacteriophages which were developed in order to be used in therapy of bacterial infections are described in the US 5 811 093. According to this document bacteriophages are developed by serial passage or by genetic engineering to obtain bacteriophages capable of delaying inactivation by any component of the animal's host defense system against foreign bodies. These phages were able to survive for longer periods of time in the circulation and the tissues than the wild-type phage.
  • the US 6 121 036 a bacteriophage preparation is described whereby the preparation consists essentially of two or more bacteriophages and these bacteriophages are isolated against different strains of bacterial organisms.
  • the US 5 366 882 A relates to isolated DNA coding for a restriction endonuclease and/or methy- lase in order to provide a system for the use in various genetic engineering methods.
  • the SU 1 463 759 A discloses a plasmid comprising a gene for an active rexAB which limits the growth of the natural forms of T4, T5 and T7 bacteria phages as well as defective genes of repressors cl and cro which suppress rexAB expression. These plasmides increase the resistance of E.coli cells to various bacteria phages .
  • the SU 1 130 602 A relates to a recombinant plasmid DNA which determines the synthesis of endonuclease of restriction Eco RV.
  • the WO 92/01791 Al relates to a modified bacterium comprising the gene of a lytically active membrane protein from bacteriophages or a gene of a lytically active toxin-release gene in order to produce bacteria ghosts which can be used as immunogenes .
  • the EP 0 278 697 A2 discloses an expression system comprising a kil gene which will be used to release paraplasmic proteins into the culture medium in order to produce proteins of medical and industrial importance.
  • phages induced host cell lysis, in Gram+ and Gram- bacteria which results in a massive release of cytoplasmic and cell wall components including endotoxin or superantigenic exotoxin, lipoteichoic acids and mu- ropeptides of bacteria.
  • cytoplasmic and cell wall components including endotoxin or superantigenic exotoxin, lipoteichoic acids and mu- ropeptides of bacteria.
  • the object of the present invention is therefore, to provide phages which can be used in phage therapy to effectively kill specific bacteria and simultaneously avoiding undesired side effects, e.g. release of cell debris.
  • the object of the present invention is to provide phages which are safe and will not cause • any harm to the organism to which they are administered, which will, however, be effective in killing any strain of bacteria- and therefore also kill bacteria which are resistant to antibiotics .
  • the object of the present invention is to provide phages which can replace antibiotics in antibacterial therapy.
  • the object of the present invention is achieved by a phage characterized in that its lysis cassette comprises a non-lytic modification and a non essential region of its genome comprises at least one kil-gene.
  • lysis cassette is used for the sequence in the phage genome which is necessary for cell lysis upon phage infection.
  • bacteriophages which comprise a lysis cassette are either virulent phages or temperent phages, e.g. phages which have the ability to carry out a lytic cyclus once they have infected the bacterial cells.
  • the lysis cassette therefore comprises in particular genes coding for proteins necessary to cause cell lysis.
  • the lysis cassette according to the present invention also comprises regions of the phage genome which are able to regulate the expression and activity, respectively, of these genes the products of which are capable of causing cell lysis.
  • lysis cassettes may comprise but are not limited to lysozymes, transglycosylases, endopeptidases, amidases (see Young R. (1992) Bacteriophage lysis: mechanism and regulation. Microbiol. Rev.56, 430-481) as well as hol-genes, lysis gene L of the male-specific RNA phage MS2 , 2 maturation protein from group III RNA phage Q ⁇ , lysis gene E of single stranded DNA phage 0X174 and others. These genes and lysis systems have been well characterised and the person skilled in the art will be able to define the lysis cassette of a given phage without undue burden.
  • non-lytic modification in the scope of the present invention comprises any modification in the above mentioned lysis cassette which modification renders a phage comprising a lysis cassette non-lytic.
  • a phage with a non-lytic modification in its lysis cassette does not cause cell lysis once it has infected a bacterial cell.
  • This non-lytic modification comprises in particu- iQ rr tiTJ ⁇ -3 w if ⁇ - O ffi s; ⁇ ffi Hi rt TJ r
  • O O UD ⁇ i ⁇ ⁇ P ⁇ 0 ) TJ ⁇ ⁇ - rt ⁇ H w h- i -o rt 1-J 1 o ⁇ ⁇ - H 3 h- o
  • ⁇ ⁇ - K; ⁇ P ⁇ - ⁇ Hi ⁇ ⁇ Oi • s; if ⁇ o ⁇ P ⁇ Hi o ⁇ - ⁇ - ⁇ P CO en if ' ⁇ - P co ⁇ ⁇ - TJ ⁇ O ii o CO P. f- H- rt P a ⁇ P CO ⁇ ⁇ 0 ) ⁇ ⁇ P ⁇ ⁇ o n P J ⁇ > iQ P 0 ) rt 1 rt * P 1 ⁇ ⁇ - rt i: ' ⁇ s, rr- O ⁇ - ⁇ - ⁇ - ti ⁇ ⁇ if ? CO ⁇ - ⁇ - Hi O 3 rt ⁇ ro rt
  • any other system can be used.
  • a system in which the kil-gene is selected to kill selected bacteria will not kill the host cells in which it will be propagated.
  • a further example of a kil-gene is the holin-gene:
  • the function of phage encoded holins is to punch a hole in the inner membrane which leads to efficient cell killing without an immediate disintegration of the cell.
  • holins of different phages are active in Gram-negative as well . as in Gram-positive bacteria.
  • the propagation of the phage comprising a holin gene as a kil-gene may be carried out in bacteria which comprise anti- sense RNA in order to completely silence the holin gene during propagation of the modified phage ⁇ (see Blasi et al . FEMS Micro- ' biol. Rev. 17 (1995) 191-205 which is incorporated herein by reference) .
  • 'a kil-gene relates to type II restriction/modification systems.
  • the action of the restriction endonu- clease is prevented in the host used for propagation of the modified phage through modification of the substrate by a corresponding methylase.
  • non-essential region is a sequence of the genome which is not essential for " the phage which means that a phage comprising the kil-gene is still able to infect cells and propagate normally.
  • the person skilled in the art will be able to detect and select such a non-essential region in a phage genome without undue burden since such non-essential regions are already described in the literature and have been used for insertion of foreign genes and a person skilled in the art will be able to find further non-essential regions in a given phage.
  • the phage according to the present invention is therefore able to infect bacterial cells, propagate within these cells and cause a loss of viability to the bacterial cells, however, without causing cell lysis. Therefore, phages are provided which kill bacteria, however, without the usual side effects caused by cell debris. These phages are therefore efficient and safe for therapeutical use.
  • any phage which is able to cause cell lysis can be modified according to the present invention.
  • the phage may be selected according to the specific bacteria to be killed.
  • phages By using a peptide library of one or more bacteria strains phages with a broad host strain spectrum can be provided. In particular such which can infect the majority of clinical isolates, e.g. of Staphylococcus aureus isolates. Thereby, the problem of phage resistant bacterial mutants can be solved.
  • the non-lytic modification in the cell lysis cassette comprises an expression-disrupting modification in a holin gene.
  • the holin gene is the gene coding for the holin protein which is a small protein which couses a non-specific lesion in the cytoplasmic mebrane which allows transit of the endolysin (see below) to the cell wall which is accompanied by cell lysis.
  • the holin gene is therefore one of the main factors contributing to cell lysis.
  • the expressed holin protein is either modified in such a way to render it inactive or the holin protein is not expressed at all.
  • the modification may be any mutation in the gene or in a DNA region necessary for the holin-regulation or in the above mentioned region, but also a ,deletion of part of the gene or of the complete gene. Phages with an expression-disrupting modification in the holin gene do not cause lesions in the cytoplasmic membrane of the bacteria. Thereby, such phages are not able to cause cell lysis.
  • type I ⁇ S, P22 gpl3, P2 Y, Pi LydA, PRDl ORFM
  • type II 21 S, PA-2 S, 080 S, T7 gpl7,5, T3 Lys , and Hpl ORF78
  • type III solely occupied by T4 t, which does not resemble any other holin sequence.
  • gene families which have clear evolutionary relationships discernible from sequence similarity (FEMS Microbiology Reviews 17 (1995) 191-205) .
  • any other here not mentioned holin-gene will be comprised by the present application.
  • a further preferred embodiment of the phage according to the present invention is characterized in that the non-lytic modification in the cell lysis cassette comprises an expression- disrupting modification in an endolysin gene.
  • the endolysin is a protein whose substrate is the peptidoglycan of the bacteria. Also here the same as described for the holin gene is applied: By entering an expression-disrupting modification in the endolysin gene of the phage the phage is unable to lyse the cell.
  • T4 lysozyme has been shown to be a soluble, monomeric 15-kDa protein with a basic isoelectric point. Because of the absence of a signal sequence, T4 lysozyme, and the other endolysins as well, accumulate in an active, fully folded form in the cytoplasm during the vegetative cycle. At the end of the vegetative cycle, and in fact defining the end of the vegetative cycle, the holin protein acts to allow release of the accumulated endolysin to the cell wall. Rapid degradation of the peptidoglycan ensues and macroscopic lysis occurs, usually in a characteristically abrupt fashion (FEMS Microbiology Reviews 17 (1995) 191-205) .
  • the holin and endolysin gene can be identified in any phage by sequence gazing and verification with a functional test: Computer programs can be used which identify putative endolysins based on their hormology with known and characterized phage endolysins. Holin genes may be identified based on their common architecture which features transmembrane domains sepa- rated by ⁇ -turns as proposed by Steiner et al . (1993) J. Baceriol. 175:1038-1042, which is incorporated herein by reference .
  • the kil-gene is a gene which codes for a nuclease.
  • phages are provided which attack the DNA and RNA, respectively, of the bacterial cell thereby rendering the bacteria non-viable.
  • the kil-gene is a gene which codes for a restric- tionendonuclease.
  • the restrictionendonuclease is an enzyme which recognizes a specific sequence in a DNA and which cuts the DNA at the recognized region.
  • Many restrictionendonucleases have been identified, whereby an endonuclease is specific for one bacteria, which means that it recognizes DNA which does not- naturally occur in the bacteria and thereby digests this foreign DNA. Of course it is necessary that the enzyme does not recognize the phage DNA.
  • the kil-gene is a gene which codes for a toxin.
  • the term "toxin” comprises any product which poisens the cell (metabolism) .
  • Such toxin genes may be derived from proteic plasmid stabilizations systems, e.g. the doc gene of P, as well as of other post segregational killing systems, e.g. hok of plasmid Rl or gef of E. coli 12 (Poulsen L.K., arsen N. . , Molin S. and Ander ⁇ son P. (1984) Molec. Micro- biol.. 3:1463-1472; Jensen R.B. and Gerdes K. (1995), Molec. Mi- crobiol . 17:205-210) .
  • Many other toxins are well known in the art and any suitable toxin may be applied which attacks the bacteria.
  • the phage comprises a non-replicative modification in its genome.
  • non-replicative modification comprises any modification in the phage genome whereby such a modified phage does not replicate.
  • the modification may be any mutation or deletion as described above in a sequence of the genome which is necessary for the phage replication, e.g. a gene which codes for a protein necessary for replication or a sequence in the genome which regulates the replication of the phage.
  • a phage is provided which infects bacterial cells, causes cell death without cell lysis, however, which does not replicate and therefore is biologically contained. Thereby a very safe system is provided for killing bacteria without causing danger to other bacterial cells in the environment.
  • a non-replicative modification is present in at least one of the phages genes which code for proteins required for DNA replication.
  • a non-replicative modification is present in at least one of the phages genes which code for proteins required for DNA replication.
  • ⁇ the non-replicative modification is present in at least one of the phages genes which code for proteins required for phage maturation.
  • the phage DNA is replicated.
  • one of the proteins required for phage maturation is not expressed. Thereby, parts of phages are produced in the bacterial cell, however mature phages are not produced so that no further infection by the phages will occur.
  • the non-replicative modification is present in at least one of its genes which code for coat/capsid proteins. These proteins are necessary for the assembly to a mature phage. If these proteins are not present a mature phage will not be produced whereby such phages comprising the non-replicative modification in a coat/capsid gene will infect a bacterial cell however will not propagate.
  • a further aspect of the present invention is a method for producing a phage as described above, whereby
  • a kil-gene is entered into a non-essential region of the phage genome
  • the modified phage genome is entered into cells of a host strain
  • the modification of the lysis cassette can be achieved by any known method for entering mutations in genes, e.g. site directed mutation.
  • the respective genes can be identified as mentioned above by sequence gazing and verification by a functional test.
  • the modified phage genome is entered into a host strain i order to propagate. Also these methods are well known in the state of the art and any appropriate method may be applied, e.g. electro- poration, transfections with liposomes, CaCl 2 etc.
  • the host strain which will preferably be an appropriate bacterial cell
  • phages will propagate comprising the modified genome. These phages are then isolated and purified from the bacteria and can be stored appropriately according to methods well known in the art, e.g. at 4°C or lyophilized depending on the storage time,.
  • a gene coding for a restrictionendonuclease is entered into the phage genome and the modified phage genome is entered into cells of a host strain which expresses the corresponding methylase.
  • restrictoendonuclea ⁇ es are specific to a given species of bacteria and recognize DNA which is foreign to the bacterium.
  • the method of recognition is due to a bacterium specific methylase which is an enzyme which methy- lates the bacteria DNA in a way which is specific to that bacteria. Any DNA which enters the ' bacteria does usually not comprise this specific methylation and is therefore recognized by the restrictionendonuclease also specific for that bacteria.
  • a phage comprising a gene coding for a restrictionendonuclease can be entered into any host strain which expresses the corresponding methylase without killing the host strain, since the restrictionendonuclease will not recognize the host strain DNA as foreign DNA and therefore not digest it.
  • a gene which codes for the corresponding methylase is cloned into the cells of the host strain. Methods for cloning genes into cells are well known in the art. By cloning the methylase gene into the host strain, any host strain can be used for the propagation of the modified phage since the restrictionendonuclease will not recognize the host strain DNA as foreign DNA and therefore not digest it. With this method according to the present invention it is possible to design any specific phage- host strain system.
  • the modified phage Since the propagation conditions as described above do not impose any selective pressure on the phage containing the restrictionendonuclease gene the modified phage is maintained genetically stable over many, e.g more than 5, preferably 10, most preferred 20, generations. This can otherwise be a problem when the modified phage is equiped with the kil-gene, the expression of which is not tightly controlled. Since the selective pressure operating during the propagation will generate less effective progeny.
  • the phage genome is non-replicatively modified.
  • non-repli- catively modified is the same as defined above for the "non-replicative modification” whereby methods for modifying DNA, e.g. mutating and deleting DNA, respectively, are well known in the art.
  • the non-replicative modification is carried out by expression-disruptingly modifying at least one of the phages genes which code for proteins required for DNA replication and for phage maturation, respectively.
  • the gene for phage maturation a gene which codes for a coat/capsid protein is modified.
  • the wild-type gene which corresponds to the expression-disruptingly modified gene in the phage genome is cloned into the host strain.
  • a host strain which comprises the gene which is modified in the phage genome. If the non-replicative phage genome is entered into such a host strain comprising the wild-type gene the phage is able to replicate in
  • phage DNA comprises the phage genome. However, it may also comprise only a part of the phage genome which is necessary in order to produce phages according to the ' present invention. Also the phage DNA may be one or more plas- mides . The phage DNA may comprise the phage genes in an order in which it naturally occurs in phages. However, the phage genes may also be cloned into a plasmid in a different non-natural order. Furthermore, the phage DNA may comprise additional sequences and genes which do not naturally occur in the phage genome .
  • the non-lytic modification in the lysis cassette comprises an expression-disrupting modification, in a holin gene.
  • non-lytic modification in the lysis cassette comprises an expression-disrupting modification in an endolysin gene.
  • the kil-gene is a gene which codes for a nuclease.
  • the kil-gene is a gene which codes for a restrictionendonuclease.
  • the kil-gene is a gene which codes for a toxin.
  • the phage DNA comprises a non-replicative modification.
  • the non replicative modification is present in at least one of its genes which code for proteins required for DNA replication, in at least one of its genes which code for proteins required for phage maturation, and in at least one of its genes which code for coat/capsid proteins, respectively.
  • a further aspect of the present invention is the use of the phage as an antibacterial agent.
  • This agent can be used in organisms as well as in any non-living substance, e.g. in a selection medium for cultivation, in any liquids, creams and substances which must be free from bacteria.
  • the agent may of course comprise any further bactericidal, virucial, etc. agents, e.g. antibiotics, chemical substances, etc.
  • a pharmaceutical preparation which comprises phage according to the present invention as described above.
  • the pharmaceutical preparation may comprise any further antibacterial substance, e.g. antibiotics, virucidal substance, and others.
  • the pharmaceutical preparation further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutical preparation according to the present invention can be used as a safe treatment of bacterial infections in organisms . Since the bacterial cells do not lyse any side effects of conventional phage therapy are avoided. Furthermore, any side effects resulting from conventional antibiotic treatment are also avoided. This pharmaceutical preparation is particularly useful for the treatment of bacteria which are resistant to antibiotics. The pharmaceutical preparation may be used for example in a treatment against Staphylococcus aureus infections .
  • the present invention provides a method for treatment of bacterial infections in an organism whereby a pharmaceutical preparation according to the present invention as described above is administered.
  • fig.l shows the strategy for the production of a phage according to the present invention.
  • fig. 2 shows the destruction of bacterial cells infected with phage over a time period.
  • Example 1 Construction of a phage containing a restrictionendonuclease gene (s. Fig.l)
  • the restrictionendonuclease II gene of Bacillus subtilis RUB562 (Bacillus globigii) was amplified by PCR using chromosomal DNA.
  • the 5 'primer was designed to incorporate the riboso e binding site of the Bglll gene and to introduce a Ba HI restriction site.
  • the 3 'primer included a Hindlll-restriction site.
  • the resulting PCR fragment was cloned into the BamHI/Hindlll site of the phage vector Ml3mpl'8 under transcriptional control of the lac promoter (Ml3-BglIIR) .
  • BglllM Bglll methylase gene
  • E. coli-P. aeruginosa shuttle vector pUCP24 The Ml3-BglIIR. phage was propagated on E. coli MC4100F' containing plasmid pUCP24BglIIM in LB broth containing 30 ⁇ g/ml tetracy- cline, 15 ⁇ g/ml gentamycin and 3 M IPTG (s. A in Fig.l). Overnight cultures- were centrifuged at 6000 g for 5 min and the supernatant was passed twice through a 0.20 ⁇ m filter.
  • the progeny phage was used to infect wt bacteria (s. B in Fig.l) . Host DNA was degraded by the enzyme (s. C in Fig.l) . The bacteria remain structurally intact for several hours.
  • Example 2 Efficacy of host cell killing (s. Fig. 2)
  • the killing efficacy of the Ml3-BglIIR phage was assessed on E. coli strain MC4100F'.
  • the culture was grown in 10 ml LB broth containing 30 ⁇ g/ml tetracycline and 3 mM IPTG at 37°C.
  • the culture was grown to an OD600 of.0.2 and infected with a MOI (multiplicity of infection) of 2.
  • MOI multiplicity of infection

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Abstract

L'invention concerne un bactériophage dont la cassette de lyse comprend une modification non lytique et dont une région génomique non essentielle comporte au moins un gène kil. L'invention concerne également un procédé de production de ce bactériophage, un ADN phagique, un agent antibactérien et une préparation pharmaceutique comprenant ledit bactériophage.
PCT/EP2001/012169 2000-10-25 2001-10-22 Bacteriophage modifie comprenant une modification non lytique et exprimant un gene kil WO2002034892A1 (fr)

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EP1435788A2 (fr) * 2001-09-27 2004-07-14 Gangagen, Inc. Bacteriophages deficients en lysine a immunogenicite reduite
EP1560491A2 (fr) * 2002-11-14 2005-08-10 Gangagen, Inc. Bacteriophages a holine modifiee et leurs utilisations
WO2008086881A1 (fr) * 2007-01-15 2008-07-24 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Bactériophages génétiquement modifiés, destinés notamment à la lutte contre des procaryotes pathogènes et leur effet pathologique, leur production et leur utilisation
DE102010013834A1 (de) 2008-03-30 2010-12-16 VLB Berlin e. V. Institut für Mikrobiologie Mittel zur Blockierung der Vermehrung und der Abtötung von Bakterien
WO2019068006A1 (fr) * 2017-09-29 2019-04-04 The Charles Stark Draper Laboratory, Inc. Lyse cellulaire contrôlée par stimulus
US10676721B2 (en) 2009-03-05 2020-06-09 Trustees Of Boston University Bacteriophages expressing antimicrobial peptides and uses thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1435788A2 (fr) * 2001-09-27 2004-07-14 Gangagen, Inc. Bacteriophages deficients en lysine a immunogenicite reduite
EP1435788A4 (fr) * 2001-09-27 2009-07-08 Gangagen Inc Bacteriophages deficients en lysine a immunogenicite reduite
EP1560491A2 (fr) * 2002-11-14 2005-08-10 Gangagen, Inc. Bacteriophages a holine modifiee et leurs utilisations
EP1560491A4 (fr) * 2002-11-14 2008-04-30 Gangagen Inc Bacteriophages a holine modifiee et leurs utilisations
WO2008086881A1 (fr) * 2007-01-15 2008-07-24 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Bactériophages génétiquement modifiés, destinés notamment à la lutte contre des procaryotes pathogènes et leur effet pathologique, leur production et leur utilisation
DE102010013834A1 (de) 2008-03-30 2010-12-16 VLB Berlin e. V. Institut für Mikrobiologie Mittel zur Blockierung der Vermehrung und der Abtötung von Bakterien
US10676721B2 (en) 2009-03-05 2020-06-09 Trustees Of Boston University Bacteriophages expressing antimicrobial peptides and uses thereof
WO2019068006A1 (fr) * 2017-09-29 2019-04-04 The Charles Stark Draper Laboratory, Inc. Lyse cellulaire contrôlée par stimulus

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