(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`BS
`
`I lllll llllllll II llllll lllll lllll lllll llll I II Ill lllll lllll lllll 111111111111111111111111111111111
`
`(10) International Publication Number
`WO 2008/075949 Al
`Willem, Meindert [NL/NL]; Oude Bennekomseweg 77,
`NL-6717 LM Ede (NL).
`
`(74) Agent: VAN WESTENBRUGGE, Andries; Postbus
`29720, NL-2502 LS Den Haag (NL).
`
`(81) Designated States (unless otheiwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, CH,
`CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG,
`ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL,
`IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK,
`LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW,
`MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL,
`PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV, SY,
`Tl, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA,
`ZM, ZW.
`
`(84) Designated States (unless otheiwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
`FR, GB, GR, HU, IE, IS, IT, LT, LU, LV, MC, MT, NL, PL,
`PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM,
`GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`with international search report
`with sequence listing part of description published sepa(cid:173)
`rately in electronic Jann and available upon request from
`the International Bureau
`
`(19) World Intellectual Property Organization
`International Bureau
`
`( 43) International Publication Date
`26 June 2008 (26.06.2008)
`
`PCT
`
`(51) International Patent Classification:
`A61P 1100 (2006.01)
`A61P 3110 (2006.01)
`A61P 3104 (2006.01)
`A61P 1114 (2006.01)
`A61P 9100 (2006.01)
`A61P 37100 (2006.01)
`A61P 9110 (2006.01)
`A61K 35174 (2006.01)
`A61P 3108 (2006.01)
`
`(21) International Application Number:
`PCT/NL2007 /050667
`
`(22) International Filing Date:
`18 December 2007 (18.12.2007)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`06126673.0
`
`20 December 2006 (20.12.2006) EP
`
`(71) Applicant (for all designated States except US): STICHT(cid:173)
`ING TOP INSTITUTE FOOD AND NUTRITION
`[NL/NL]; Nieuwe Kanaal 9A, NL-6709 PA Wageningen
`(NL).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): TROOST, Fred(cid:173)
`erik, Jan [NL/NL] (NL). KLEEREBEZEM, JWichiel
`[NL/NL]; Indira Ghandisingel 4, 6716 GG Ede, NL-6716
`GG Ede (NL). BRUMMER, Robert-Jan [NL/NL]; Kum(cid:173)
`menaedestraat 72, NL-6165 BX Geleen (NL). DE VOS,
`
`---iiiiiiiiiiiiiii
`iiiiiiiiiiiiiii -iiiiiiiiiiiiiii
`iiiiiiiiiiiiiii -iiiiiiiiiiiiiii ---
`----iiiiiiiiiiiiiii
`iiiiiiiiiiiiiii ----
`
`0--..
`"'1"
`
`0--.. "' r-
`
`--. ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
`0
`~ (54) Title: MODULATION OF HUMAN MICROSOMAL TRIGLYCERIDE TRANSFER PROTEIN (~ITP OR MTTP) GENE
`0 EXPRESSION BY FOOD-GRADE/INGESTED DIETARY MICROORGANISMS
`M O (57) Abstract: The present invention relates to the field of using microorganisms, especially food grade bacteria, to modulate in(cid:173)
`> testinal MTP expression levels in order to treat and/or prevent weight gain, obesity, atherosclerosis, hyperglyceridaemia, hyper(cid:173)
`
`~ cholesterolaemia, diabetes, dyslipidaemia and/or disorders associated with impaired intestinal immune response to antigens.
`
`1 of 47
`
`PENN EX. 2293
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`WO 2008/075949
`
`PCT/NL2007 /050667
`
`Modulation of human microsomal triglyceride transfer protein (MTP or MTTP) gene
`
`expression by food-grade/ingested dietary microorganisms
`
`5
`
`FIELD OF THE INVENTION
`
`The present invention relates to the field of food-grade microorganisms, especially
`
`bacteria and/or their components. Especially, the use of food-grade microorganisms,
`
`which are capable of modulating intestinal MTP gene expression, for the preparation of
`
`food or feed compositions, food or feed supplements or pharmaceutical compositions
`
`10
`
`for the treatment and/or prevention of a sub-optimal (or non-healthy) intestinal
`
`microbiota,
`
`weight
`
`gam,
`
`obesity,
`
`atherosclerosis,
`
`hyperglyceridaemia,
`
`hypercholesterolaemia, diabetes, dyslipidaemia and/or disorders associated with
`
`impaired intestinal immune response to antigens is provided herein. Also provided are
`
`compositions comprising one or more microbial strains or components thereof (such as
`
`15
`
`cell fractions) which are capable of modulating MTP gene expression in human
`
`intestinal cells or tissues in vivo and in vitro, as are methods for isolating such strains
`
`and for establishing administration/dosage regimes for such strains. Furthermore, the
`
`use of intestinal MTP gene expression as a biomarker for the health status of humans
`
`and their associated intestinal micro biota is provided herein.
`
`20
`
`BACKGROUND OF THE INVENTION
`
`Microsomal triglyceride transfer protein (MTP), which catalyzes the transport of
`
`triglyceride, cholesterol ester and phospholipids between phospholipids surfaces, is a
`
`heterodimer, consisting of an 88 kDa catalytic domain which is non-covalently
`
`25
`
`associated with a 58 kDa PDI (protein disulfide isomerase). The human cDNA and
`
`genomic DNA encoding the large subunit MTP have been cloned and characterized
`
`(Sharp et al. 1993, Nature 365: 65-69). The human MTP gene was found to be
`
`primarily expressed in liver and intestinal tissue, which is compatible with its proposed
`
`function in triglyceride transfer (Hagan et al. 1994, J Biol Chem 269: 28737-28744).
`
`30 MTP plays a role in the assembly and secretion of apolipoprotein B (apoB) containing
`
`lipoproteins and high plasma levels of these lipoproteins may be associated with
`
`atherosclerosis and coronary heart diseases. Humans carrying non-functional MTP
`
`genes have a condition referred to as abetalipoproteinaemia and a defective production
`
`2 of 47
`
`PENN EX. 2293
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`WO 2008/075949
`
`PCT /NL2007 /050667
`
`2
`
`of apoB-containing lipoproteins. Hepatic over-express10n of MTP results in an
`
`increased in vivo secretion of VLDL (very low density lipoprotein) triglycerides and
`
`apoB (Tietge et al. 1999, J. Lipid Res. 40: 2134-2139). MTP liver-specific knock-out
`
`mice results in an abrogation of VLDL/LDL production (Chang et al. 1999, J Biol
`
`5
`
`Chern 274: 6051-6055). Similarly, it was shown that the inhibition of MTP activity
`
`causes a decrease in the secretion rate of apoB-containing lipoproteins in human and
`
`intestinal cells in vitro (Jamil et al. 1996, PNAS 93: 11991-11995; Van Greevenbroek
`
`et al. 1998, J Lipid Res 39: 173-185). These findings, therefore, suggest that MTP plays
`
`a role in modulating lipoprotein production in the liver and intestine.
`
`10
`
`Recently, MTP was found to be also involved in modulating the intestinal immune
`
`response to antigens. Lipid antigens are presented to T cells by CD 1 molecules, which
`
`are major histocarnpatibility complex (MHC) class I-hornologues. CDl is expressed on
`
`myeloid cells, hepatocytes and intestinal epithelial cells. The type I CDl molecules
`
`15
`
`(CDla, CDlb and CDlc) are expressed on dendritic cells in the intestinal mucosa. It
`
`was shown previously that MTP regulates CD 1 d function and, hence, natural killer T
`
`(NKT) cell biogenesis (Brozovic et al. 2004, Nature Med 10: 5: 535-9). Recent
`
`observations that MTP is involved in antigen presentation, and is able to regulate
`
`CDla, CDlb and CDlc production, suggest that MTP is important in the host response
`
`20
`
`to microbial pathogens. The presence of type 1 CDl molecules on dendritic cells
`
`indicates a putative role of MTP in the pathogenesis of rnucosal inflammation-related
`
`disorders (Kaser A, Hava D, Yoshida M, Kuo T, Nagaishi T, Dougan S, Lugt Vander
`
`B, Haddad W, Brenner M, Blumberg R. Microsomal triglyceride transer protein
`
`regulates endogenous and exogenous antigen presentation by group 1 Cdl molecules.
`
`25
`
`Gastroenterology 2006;130: 4 suppl 2; 126). Furthermore, recent data suggest that MTP
`
`regulates an intestinal barrier-protective CDld pathway that is mediated by NKT cells
`
`(Kaser A, Yoshida M, Furuta G, Zhu F, Davidson N, Colgan S, Blumberg R. Intestinal
`
`Microsomal Triglyceride Transfer Protein (MTP) regulates Cdld function on the
`
`intestinal epithelium and protects
`
`from mortality
`
`in
`
`the oxazolone model.
`
`30 Gastroenterology 2006;130: 4 suppl 2; 126).
`
`The regulation of MTP gene expression seems to be complex. In hamsters, high fat and
`
`high cholesterol diets have been shown to up-regulate hepatic MTP mRNA levels. In
`
`3 of 47
`
`PENN EX. 2293
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`WO 2008/075949
`
`PCT /NL2007 /050667
`
`3
`
`vitro studies on human hepatoma cells have shown that ethanol (Lin et al. 1997,
`
`FASEB J. 11, 1145-1152), fresh garlic (Lin et al. 2002, J Am Soc Nutr Sci, 132: 1165-
`
`1168) and insulin (Lin et al. 1995, J Lipid Res 36: 1073-1081) down-regulate hepatic
`
`MTP expression.
`
`5
`
`Some drugs, such as diaminoindanes and benimidazole-based compounds, with MTP(cid:173)
`
`inhibitory activity are under investigation for the treatment of hyperlipidemia (Burnett
`
`JR 2006, !Drugs, Jul;9(7):495 -9; Chandler CE, Wilder DE, Pettini JL, Savoy YE,
`
`Petras SF, Chang G, Vincent J, Harwood HJ Jr, J Lipid Res. 2003 44(10):1887-901).
`
`10
`
`These drugs mostly inhibit both liver and the intestinal MTP expression and may result
`
`in fatty livers. Intestinal-specific MTP inhibitors would be desirable, preferably ones
`
`which are based on natural food-grade products. Fresh garlic seems to contain MTP
`
`inhibiting substances which are relatively specific for inhibiting intestinal MTP
`
`expression in rats, 3 hours after oral administration (Lin MC, Wang EJ, Lee C, Chin
`
`15
`
`KT, Liu D, Chiu JF, Kung HF, J Nutr. 2002 Jun;l32(6):1165-8).
`
`However, the components, which are responsible for this effect, have not yet been
`
`identified and it is not clear whether the effect and specificity would be the same in
`
`humans. Furthermore, the addition of large quantities of fresh garlic to food products
`
`would limit the applicability to certain types of food.
`
`20
`
`It is, therefore, an object of the invention to provide alternative, food grade MTP(cid:173)
`
`expression modulators, especially MTP gene expression inhibitors and activators,
`
`methods for identifying and isolating these and compositions comprising these. It is a
`
`further object of the invention to provide a method for evaluating and monitoring the
`
`25
`
`health status of the human intestinal microbiota, and means for distinguishing between
`
`a healthy intestinal microbiota and an unhealthy or sub-optimally healthy intestinal
`
`condition. In addition, means for treating and/or preventing a sub-optimal or abnormal
`
`intestinal microbiota are provided, whereby a sufficient amount of a MTP-gene
`
`expression modulating composition, comprising or consisting of at least one
`
`30 microorganism capable of modulating intestinal MTP-gene expression, is administered.
`
`GENERAL DEFINITIONS
`
`4 of 47
`
`PENN EX. 2293
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`WO 2008/075949
`
`PCT /NL2007 /050667
`
`4
`
`"Lactic acid bacteria" and "lactic acid producing bacteria", 1s used herein
`
`interchangeably and refers to bacteria, which produce lactic acid as an end product of
`
`fermentation, such as, but not limited to, bacteria of the genus Lactobacillus,
`
`Streptococcus, Lactococcus, Oenococcus, Leuconostoc, Pediococcus, Carnobacterium,
`
`5
`
`Enterococcus. In addition, Bifidobacterium and Propionibacterium species are
`
`considered for this application to belong to lactic acid bacteria although they have a
`
`distinct phylogenetic position.
`
`"Probiotics" or "probiotic strain(s)" refers to strains of live or viable micro-organisms,
`
`preferably bacteria, which when administered in adequate amounts provide a health
`
`10
`
`benefit to the host subject, e.g. when ingested (e.g. orally, enterally or by inhalation) by
`
`a subject. Probiotics are defined as "viable microbial food supplements which, when
`
`taken in the right doses beneficially influence human health" (Salminen et al. 1998,
`
`WH02002).
`
`"Micro-organisms" include bacteria and fungi, such as yeasts. When reference herein is
`
`15 made to bacteria, it is understood that the embodiments also apply to other
`.
`.
`microorgamsms.
`
`"Enteral" refers herein to the delivery directly into the gastrointestinal tract of a subject
`
`(e.g. orally or via a tube, catheter, capsules or stoma).
`
`"Food-grade" micro-organisms are in particular organisms, which are considered as not
`
`20
`
`harmful, when ingested by a human or animal subject.
`
`"Components" of microorganisms or "inactivated" microorganisms refers to non-viable
`
`microorganisms, such as dead cells, cell fragments, and the like.
`
`A "subject" refers herein to a human or animal, in particular a vertebrate, such as but
`
`not limited to domestic animals.
`
`25
`
`The term "comprising" is to be interpreted as specifying the presence of the stated
`
`parts, steps or components, but does not exclude the presence of one or more additional
`
`parts, steps or components. A composition comprising a lactic acid bacterium may thus
`
`comprise additional bacterial strains etc. However, a lactic acid bacterium or a mixture
`
`of several distinct lactic acid bacteria is preferably a main active component of a
`
`30
`
`composition of the invention. More preferably, a lactic acid bacterium or a mixture of
`
`several distinct lactic acid bacteria is the sole active component of a composition of the
`
`invention.
`
`5 of 47
`
`PENN EX. 2293
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`WO 2008/075949
`
`PCT /NL2007 /050667
`
`5
`
`"Percentage" or "average" generally refers to percentages of averages by weight, unless
`
`otherwise specified or unless it is clear that another basis is meant. For example, when
`
`referring to % gene expression, it is clear that the relative or absolute amount of a gene
`
`transcript being produced is referred to.
`
`5
`
`The term "a" or "an" does not limit to one, but is interpreted as at least one. Therefore,
`
`when reference is made to 'a bacterium' it is understood that a plurality of bacteria are
`
`encompassed.
`
`The term "derivative" refers to the biological material that represents a substantially
`
`unmodified copy of the material, such as material produced by growth of micro-
`
`10
`
`organisms, e.g. growth of bacteria in culture media. The term "derivative" also includes
`
`material created from the original micro-organism which retains the beneficial
`
`properties of the unmodified strain, but which is modified to have new additional
`
`properties, for example caused by heritable changes in the genetic material. These
`
`changes can either occur spontaneously or be the result of applied chemical and/or
`
`15
`
`physical agents (e.g. mutagenesis agents) and/or by recombinant DNA techniques as
`
`known in the art.
`
`The term "gene" means a DNA compnsmg a region (transcribed region), which is
`
`transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable
`
`regulatory regions (e.g. a promoter). A gene may thus comprise several operably linked
`
`20
`
`fragments, such as a promoter, a 5' leader sequence, a coding region and a
`
`3 'nontranslated sequence comprising a polyadenylation site. An "endogenous gene" is
`
`a gene found in its natural environment in the cells of an organism.
`
`"Expression of a gene" refers to the process wherein a DNA region which is operably
`
`linked to appropriate regulatory regions, particularly a promoter, is transcribed into an
`
`25
`
`RNA, which is biologically active, i.e. which is capable of being translated into a
`
`biologically active protein or peptide.
`
`A "transcription regulatory sequence" is herein defined as a nucleic acid sequence that
`
`is cap ab le of regulating the rate of transcription of a (coding) sequence operably linked
`
`to the transcription regulatory sequence. A transcription regulatory sequence as herein
`
`30
`
`defined will thus comprise all of the sequence elements necessary for initiation of
`
`transcription (promoter elements), for maintaining and for regulating transcription,
`
`including e.g. attenuators or enhancers. Although mostly the upstream (5 ') transcription
`
`6 of 47
`
`PENN EX. 2293
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`WO 2008/075949
`
`PCT /NL2007 /050667
`
`6
`
`regulatory sequences of a coding sequence are referred to, regulatory sequences found
`
`downstream (3 ') of a coding sequence are also encompassed by this definition.
`
`As used herein, the term "operably linked" refers to a linkage of polynucleotide
`
`elements in a functional relationship. A nucleic acid is "operably linked" when it is
`
`5
`
`placed into a functional relationship with another nucleic acid sequence. For instance, a
`
`promoter, or rather a transcription regulatory sequence, is operably linked to a coding
`
`sequence if it affects the transcription of the coding sequence. Operably linked means
`
`that the DNA sequences being linked are typically contiguous and, where necessary to
`
`join two protein encoding regions, contiguous and in reading frame.
`
`10
`
`"Modulation" of gene expression refers to an up-regulation or down-regulation of gene
`
`expression relative to one or more control subjects or control samples. It is understood
`
`that the up-regulation or down-regulation should be statistically significant, taking
`
`account of normal variation in expression levels.
`
`The term "induced" or "up-regulated" gene expression as used herein refers to the
`
`15
`
`induction of activity of a transcription regulatory sequence (especially a promoter) and
`
`the resulting increase in mRNA transcription of the nucleic acid sequence operably
`
`linked thereto. Thus, in particular, an initiation or increase in mRNA transcript
`
`production of a DNA sequence operably linked to the transcription regulatory sequence
`
`is referred to. Induction or up-regulation refers therefore to a change from no mRNA
`
`20
`
`transcription to mRNA transcription, or to a change from a certain transcription level to
`
`a higher transcription level. In particular, an increase of at least 5%, 10%, 20%, 30%,
`
`50%, or more, of mRNA transcription after induction is encompassed in this definition.
`
`Vice versa, "inhibition" or "down-regulation" or "reduction" of gene expression as
`
`used herein refers to the reduction of activity of a transcription regulatory sequence
`
`25
`
`(especially a promoter) and the resulting reduction or inhibition in mRNA transcription
`
`of the nucleic acid sequence operably linked thereto. Thus, in particular, a down(cid:173)
`
`regulation or inhibition of mRNA transcript-production of a DNA sequence operably
`
`linked to the transcription regulatory sequence is referred to. Down-regulation or
`
`inhibition refers therefore to a change from a certain level of mRNA transcription to a
`
`30
`
`lower or no transcription level. In particular, an reduction of at least 5%, 10%, 20%,
`
`30%, 50%, or more, of mRNA transcription after inhibition is encompassed in this
`
`definition.
`
`7 of 47
`
`PENN EX. 2293
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`WO 2008/075949
`
`PCT /NL2007 /050667
`
`7
`
`"Tissue-specific down-regulation" refers to the down-regulation of transcription in a
`
`specific tissue, such as the intestinal cells.
`
`"MTP inhibitor" refers to one or more microorganisms or compositions compnsmg
`
`these (or components of these) which are capable of down-regulating or inhibiting
`
`5 MTP expression after contact of a sufficient amount of the microorganim( s) with
`
`intestinal cells or tissue
`
`(either directly or indirectly, by e.g. signal transduction
`
`pathways), for a sufficient period of time. "MTP activator" or "MTP inducer" refers to
`
`one or more microorganisms or compositions comprising these (or components of
`
`these) which are capable of up-regulating MTP expression after contact of a sufficient
`
`10
`
`amount with intestinal cells or tissue for a sufficient period of time.
`
`A "sufficient period" refers to an exposure time (i.e. contact with intestinal cells) which
`
`is sufficient to result in the upregulation or downregulation of MTP gene expression.
`
`Routine experimentation can be used to determine a sufficient amount and a sufficient
`
`exposure period to achieve the desired modulation of MTP expression.
`
`15
`
`"Tissue-specific MTP inhibitor" refers to microorganisms or compositions which are
`
`capable of down-regulating or inhibiting MTP expression in a specific tissue or cell
`
`type. Vice versa, "tissue specific MTP activator" or "MTP inducer" refers to
`
`microorganisms or compositions which are capable of inducing MTP expression in a
`
`specific tissue or cell type.
`
`20
`
`"Stringent hybridization conditions" can also be used to identify nucleotide sequences,
`
`which are substantially identical to a given nucleotide sequence (such as a probe).
`
`Stringent conditions are sequence dependent and will be different in different
`
`circumstances. Generally, stringent conditions are selected to be about 5°C lower than
`
`the thermal melting point (Tm) for the specific sequences at a defined ionic strength
`
`25
`
`and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50%
`
`of the target sequence hybridizes to a perfectly matched probe. Typically stringent
`
`conditions will be chosen in which the salt concentration is about 0.02 molar at pH 7
`
`and the temperature is at least 60°C. Lowering the salt concentration and/or increasing
`
`the
`
`temperature mcreases
`
`stringency. Stringent
`
`conditions
`
`for RNA-DNA
`
`30
`
`hybridizations (Northern blots using a probe of e.g. 1 OOnt) are for example those which
`
`include at least one wash in 0.2X SSC at 63°C for 20min, or equivalent conditions.
`
`Stringent conditions for DNA-DNA hybridization (Southern blots using a probe of e.g.
`
`lOOnt) are for example those which include at least one wash (usually 2) in 0.2X SSC
`
`8 of 47
`
`PENN EX. 2293
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`WO 2008/075949
`
`PCT /NL2007 /050667
`
`8
`
`at a temperature of at least 50°C, usually about 55°C, for 20 mm, or equivalent
`
`conditions.
`
`The term "substantially identical", "substantial identity" or "essentially similar" or
`
`"essential similarity" or "variant" means that two peptide or two nucleotide sequences,
`
`5 when optimally aligned, such as by the programs GAP or BESTFIT using default
`
`parameters, share at least about 80 percent sequence identity, preferably at least about
`
`90 percent sequence identity, more preferably at least 95 percent sequence identity or
`
`more (e.g., 99 percent sequence identity). GAP uses the Needleman and Wunsch global
`
`alignment algorithm to align two sequences over their entire length, maximizing the
`
`10
`
`number of matches and minimizes the number of gaps. Generally, the GAP default
`
`parameters are used, with a gap creation penalty = 50 (nucleotides) I 8 (amino acids
`
`sequence) and gap extension penalty= 3 (nucleotides) I 2 (amino acid sequence). For
`
`nucleotides the default scoring matrix used is nwsgapdna and for proteins the default
`
`scoring matrix is Blosum62 (Henikoff & Henikoff, 1992). When two sequences are of
`
`15
`
`similar length, they are preferably optimally aligned using a global alignment algorithm
`
`(Needleman and Wunsch), while when sequences are of different lengths a local
`
`alignment algorithm is preferred (Smith and Waterman; using a Gap creation penalty of
`
`10.0 and a gap extension penalty of 0.5, and the default scoring matrix as above), in
`
`order to determine sequence identity. It is clear than when RNA sequences are said to
`
`20
`
`be essentially similar or have a certain degree of sequence identity with DNA
`
`sequences, thymine (T) in the DNA sequence is considered equal to uracil (U) in the
`
`RNA sequence.
`
`DETAILED DESCRIPTION
`
`25
`
`The present inventors found that administration of certain probiotic bacterial species
`
`(e.g. L. plantarum strain WCFSl) directly into the small intestinal lumen of human
`
`healthy subjects in vivo, resulted in significant modulation of gene expression of a large
`
`number of genes in small intestinal mucosa. Microarray analysis of a short term
`
`exposure study and a long term exposure study showed that the expression of one gene
`
`30
`
`in particular, encoding human microsomal triglyceride transfer protein (MTP), was
`
`down-regulated in mucosal biopsies taken after 1 hour exposure to the bacterial strain
`
`(short term exposure), while it wasup-regulated in mucosa! biopsies taken after 6 hours
`
`exposure to the bacterial strain (long term exposure). This was confirmed by the
`
`9 of 47
`
`PENN EX. 2293
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`WO 2008/075949
`
`PCT /NL2007 /050667
`
`9
`
`proteome analyses on the samples of the 6-hours exposure study. These analyses
`
`showed that two proteins differed consistently in all volunteers. One of these protein
`
`spots was identified as human microsomal triglyceride transfer protein (MTP), while
`
`this analysis failed to allow identification of the second spot. These two studies showed
`
`5
`
`that MTP mRNA levels were not only modulated (a decrease followed by an increase)
`
`by contact of intestinal cells with the bacteria, but that MTP protein levels were
`
`modulated (a decrease followed by an increase) as well.
`
`In a third intervention study, the investigators showed that intermittent oral intake of
`
`10
`
`the bacterial strain for 6 h increased the expression of the MTP gene in the same order
`
`of magnitude as observed in the 6-h intraluminal exposure experiment. This study thus
`
`confirmed that ingestion of live or viable food-grade bacteria and exposure of the
`
`intestinal cells or tissues to these bacteria for a sufficient period of time leads to a
`
`significant modulation ofMTP expression and MTP protein levels in the intestine.
`
`15
`
`It came as a surprise that administered intestinal bacteria were capable of significantly
`
`and consistently modulating MTP expression levels in intestinal tissue, as MTP
`
`expression was thought to be regulated by dietary compounds such as fat and
`
`cholesterol.
`
`In the third intervention study, also non-viable (killed by heating), and mid-log
`
`20
`
`harvested bacteria were administered orally at 30-min intervals to healthy volunteers
`
`during a 6-h period. While intake of the stationary phase cells of the probiotic strain
`
`resulted in a significantly increased expression of the MTP gene by 13.9 % (P = 0.179),
`
`the inventors did not observe an effect of the orally ingested non-viable cells. For the
`
`mid-log harvested live bacteria an increase in MTP expression of 6.8 % was observed,
`
`25
`
`but the investigators do not consider this minor change to have a biological
`
`significance.
`
`It is, therefore, a preferred embodiment to use food grade, live and/or viable
`
`microorganisms in the methods and products of the invention. It is another preferred
`
`embodiment to use stationary phase cultures of the microorganisms. However, neither
`
`30
`
`is essential, as for other microorganisms (e.g. other food-grade bacterial genera, species
`
`or strains) it may be feasible to use dead I non-viable cells and/or cell components as
`
`modulators of MTP expression and/or to use a different growth phases of the
`
`microorganism(s) (e.g. mid-log phase or late log-phase). The testing of which
`
`10 of 47
`
`PENN EX. 2293
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`WO 2008/075949
`
`PCT /NL2007 /050667
`
`10
`
`microorganisms or combinations thereof (which genera, species or strains, which
`
`growth phase and/or live or viable versus dead or non-viable) provide the desired effect
`
`(up-regulation or do\\rn-regulation)
`
`on MTP
`
`expression
`
`involves
`
`routine
`
`experimentation and can be carried out using undue efforts, as described herein.
`
`5
`
`The finding that live or viable microorganisms are capable of modulating intestinal
`
`MTP expression allowed the inventors to prepare food, feed or pharmaceutical
`
`compositions, comprising or consisting of at least one microorganism which is capable
`
`of modulating MTP-gene expression. Such compositions are suitable for intestinal
`
`10 MTP gene activation or inhibition, depending on what kind of modulation of MTP gene
`
`expression is required. Methods for identifying the microorganism( s) which have the
`
`desired effect on MTP gene expression and for using these to make compositions
`
`having the desired effect are described further herein below.
`
`Such MTP-activator or MTP-inhibitor compositions are suitable for treating and/or
`
`15
`
`preventing a variety of diseases and disorders associated with (too) high or (too) low
`
`MTP gene expression, such as a sub-optimal intestinal microbiota (e.g. in patients
`
`having intestinal MTP-expression levels which are higher or lower than in healthy
`
`subjects),
`
`or weight
`
`gam,
`
`obesity,
`
`atherosclerosis,
`
`hyperglyceridaemia,
`
`hypercholesterolaemia, diabetes and/or dyslipidaemia (which are associated with
`
`20
`
`abnormally high MTP gene expression and would benefit from MTP-inhibitor
`
`compositions), or abetalipoproteinemia, which is associated with mutations in the MTP
`
`gene (Di Leo E, et al. Atherosclerosis 2005 180(2):311-8) and hence, with low levels of
`
`functional MTP gene express10n
`
`(and would benefit
`
`from MTP-activator
`
`compositions). In addition, subjects suffering from an impaired intestinal immune
`
`25
`
`response and/or an increased progression or severity of an impaired intestinal immune
`
`response, and/or subjects susceptible to intestinal epithelial damage would benefit from
`
`administration of an MTP activator composition.
`
`In addition, new methods for identifying microbial modulators of MTP express10n,
`
`especially MTP activators or MTP inhibitors, are provided.
`
`30
`
`11 of 47
`
`PENN EX. 2293
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`WO 2008/075949
`
`PCT /NL2007 /050667
`
`The different embodiments of the invention are linked, and features described in one
`
`embodiment are understood to also apply to the other embodiments, unless stated
`
`11
`
`otherwise.
`
`5 Uses according to the invention
`
`In one embodiment of the invention provides the use of at least one food grade
`
`microorganism, preferably a bacterium (or component thereof), capable of modulating
`
`Microsomal Triglyceride Transfer Protein (MTP) gene expression in human intestinal
`
`tissue or intestinal cells for the preparation of a composition for the treatment or
`
`10
`
`prevention
`
`of weight
`
`gam,
`
`obesity,
`
`atherosclerosis,
`
`hyperglyceridaemia,
`
`hypercholesterolaemia, diabetes, dyslipidaemia and/or disorders associated with
`
`impaired intestinal immune response to antigens.
`
`In a further embodiment, the use of at least one food grade microorganism, preferably a
`
`bacterium (or component(s) thereof), capable of modulating Microsomal Triglyceride
`
`15
`
`Transfer Protein (MTP) gene expression in human intestinal tissue or intestinal cells for
`
`the preparation of a composition for the treatment or prevention of an abnormal or sub(cid:173)
`
`optimal intestinal microbiota compared to healthy subjects.
`
`The microorganism (e.g. bacterium), or a suitable amount thereof (or a suitable amount
`
`20
`
`and type of component thereof), is capable of either upregulating or downregulating
`
`MTP gene expression in human intestinal tissue or cells in vivo and/or
`
`in vitro,
`
`following exposure of the
`
`intestinal
`
`tissue/cells
`
`to
`
`the rnicroorganism(s), or
`
`compositions comprising these, in a sufficient amount and for a sufficient period of
`
`time A short term exposure is preferably used to obtain a downregulation of MTP as
`
`25
`
`defined herein. Short term exposure preferably means approximately one hour
`
`exposure. One hour exposure is preferably carried out as described in the examples
`
`( 1.2. description of study 1 ). A long term exposure is preferably used to obtain an
`
`upregulation of MTP as defined herein. Long term exposure preferably means
`
`approximately six hour exposure. Six hour exposure is preferably carried out as
`
`30
`
`described in the examples (1.3. description of study 2 or 1.4. description of study
`
`3).Exposure of the intestinal tissue/cells to the microorganim(s) is preferably carried
`
`out by using an enteric composition or a composition which is suitable for enteric
`
`administration (via catheter or a tube). In an alternative preferred embodiment, the
`
`12 of 47
`
`PENN EX. 2293
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`WO 2008/075949
`
`PCT /NL2007 /050667
`
`12
`
`exposure of the intestinal tissue/cells to the microorganism(s) is carried out by using a
`
`composition suitable for oral administration. Such composition is preferably an enteric
`
`composition. Enteric compositions a