Note: Descriptions are shown in the official language in which they were submitted.
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W O 97/05267 1 PCT~US96/12041
MUCOSAL DELIVERY OF POLYNUCLEOTIDES
RELATED APPLICATIONS
This ~j,'claims priority to F 13~ ,' No. 601001527, filed July 26, 1996 under 35U.S.C. 119(e).
FIELD OF THE INVENTION
This invention relates to the delivery of r ' r 1~ to mucosal and neural tissues of
~. tcb.dl!c animals, and to other tissues or cells that display the 6M1 receptor. The r ' ~ ' i 't ~ thus
delivered can be i' . genes or they can operate to modulate the immune 1~, of the animal. The
- ' ' may either increase or decrease the _ ' of a ~ ' , immune response in the animal.
B~CUI:ROUND OF THE INVENTION
Mucous ' ~~ cover the exwsed surfaces of the aerodi9estive and . ~~ ~- ' tracts, the eye
S ~-.a, the inner ear, and the ducts of all exocrine glands. Due to their " ' ~ . mucous ' .
- le, ~ foreign particles and invading microbes c : :~y. These ' 5 are capable of trapping
15 and d " most of the particles and microbes they ~ ~ . In addition, mucosal tissues have a ~ '
immune system. The cells of the mucosal immune system comprise the e G ' Iymphoid tissues IMALT).
MALT cells regularly e ~ le. a vast array of foreign ~ ' I A full-scale immune response against
every foreign b~: :r thus IcuLd would render . ' such a simple and vital process as ingesting
r U; ~ ~~~ food, for example. Therefore, the mucosal immune system must have the ability to s~ ignore
20 most foreign epitopes while it mounts a vigorous response to certain others.
The ,' of acquired 11 _ of certain foreign epitopes is known as "oral tolerance" because
it was first perceived through a " ' ' s_..~iti.;~y to certain olh_.~ ;~c ' - if ingested in
large enough; 3r It is now known that this "oral tolerance" can be acquired, at least i . aDI'~ and locally,
by surG.,;_.,t exposure of MALT cells to some foreign
Many mucosal cells are chalaclcli.cd by the relative ' of r _" '' GM1 in their plasma
' _ - GM1 is a ' - ~ ' whose lipophilic ceramide portion diffuses into and with the
, ~, ' -1i~ ' bilayer of the plasma ~ ' alla' while the 'ig ' iJc-sialic acid moieties of GM1 protrude out of
the ~clldJlalla and are displayed on the cell surface. GM1 is not limited to mucosal cells alone. In fact, GM1 is
present at some level in many different cell types of the body, but most -' ' :'~ in neuronal ' . and
' anes of mucosal cells.
GM1 can act as a receptor in the plasma ' for proteins that bind its exposed :1ig --- ' iJc-sialic
acid portion. The best known GM1 binding protein is the toxin of Vibrio choler.~e, the cholera toxin (CT). CT
consists of two types of subunits. The A1 fragment of the A subunit has ADP-G' - ylaliun activity while the B
subunit has GM1-binding activity. The holotoxin is a h-clcl~ ' s ~ _ of five B subunits and one A subunit.
The structure and function of the related E. coli heat-labile ! ' ulwJIl (LT)is quite similar to that of CT.
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W 097/05267 2 PCTAUS96/12041
The ADP-M ~lali~...âctivity ând the GM1 binding of CT are both essential to the overall toxin activity of
CT. Subunits of the holotoxin, whether A or B, do not ~ . dh'~ cause the toxin reaction in vivo. In the cell, the
A subunit's ADP-r' ,' ~ activity transfers the ADP-ribosyl moiety of NAD to the a subunit of the guanine-
~; ' ! " _ protein Gs~ the I ' ~ h ' positive regulator of adenylate cyclase.
Although the present text primarily discusses the use of GM1 binding proteins, it should be noted that
"ADP-li' ,: - _ moieties" are also - . ' ' as part of the present invention as fully as if that term was used
in place of the term "GM1 binding protein," as the latter term appears herein.
There are various other ADP-~ h.,: enzymes with . ' g function. For example, the I
C..I~IUI1IA;II (LT) of E. co/iis doubly similar to CT because its A and B subunits also display ADP-I ' - ,laliùll activity
10 and GM1 binding activity"~_"c~,ti.~
Because GM1 includes and displays an ~ ' ' portion, it can also be bound by numerous lectins
and lectin-like ~' ' Also, because of the lipophilic nature of the ceramide portion, O GM1 is readily
taken up by ' _ ~s of many cell types.
CT elicits â strong reaction by the mucosal immune system. A~ , the holotoxin or the A subunit
15 alone have been used as adjuvants to stimulate a mucosal immune reaction aoainst other selected epitopes.
S, _ to many is the CGull~ fact that CTB, when coupled to a selected epitope, has a strong ability to
effect an acquired ("oral") tolerance to the delivered epitope. The B subunit alone ~rri~ delivers the coupled
epitope to the proximity of the MALT, but generally does not by itself stimulate an ' of immune
L "' The result of such a delivery without a stimulus of immunity is often acquired ' .
The dual ,' ~ of enhanced or ' ' ' mucosal immunity, together with the influence that the
mucosal immune system has on systemic immunity, have caused cûr,~ . '' interest in the '~ ', of
- ~ - ~ and methods of ll~allll~lll involving CT or CTA as adJuvants for mucosal immunity, and CTB as an
initiator and mediator of acquired tolerance.
Current methods and c - rely on covalent coupling of a desired epitope to CTB for delivery of the
25 epitope to the MALT with the purpose of gaining acquired tolerance to the epitope. This scheme has been especially
.;alud by those ~ - t:;,led in àdJ~ ~_ problems of allergy, allograft rejection, and ~ diseases.
Likewise, workers wishing to stimulate acquired mucosal, and possibly systemic, immunity have combined
their selected antigen with CT or CTA to achieve enhanced mucosal immunity.
In both cases, the instability of the delivered protein epitope and of the coupling to CTB have been
30 ' to the ~rr~l;.L~ss and duration of the desired exposure of MALT cells to the epitope. The invention
described herein add~L~es this problem in a novel way. Beyond the novelty of the solution to a ' ; , in the
current state of the art, the present invention also has great utility in gene therapy a,. '
Described herein is an invention that delivers an active PO1~ULI~U ' to target cells, such as MALT cells,
by - ' ~ _ or coupling the pol~.,uLl~olid~ to a GM1- or other mucosa-binding protein. The delivered molecule,
35 being a p~'~ '/ Ihle rather than a protein, has an effect in a single delivery event that can be many hundred- or
~ ' fold greater than the delivery of a single protein. This is because the active r-~- ~ JtiJe can direct,
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-3-
encode, or ' . ;__ affect the ultimate ~ of many copies of the desired protein. Further, it can do so for
hours or days if l:A~ ;oSO~ or on a r ~ basis if the rN~ stably _ dl~o into the
genome of the cell to which it is delivered. Similar results can not possibly be achieved when the delivered molecule
is merely a protein.
Since the invention employs binding proteins that do not only recognize mucosal cells, the invention also
has utility in the field of gene therapy. The binding protein and ' _ ' of the target cell can be
selected andlor , ' ' such that a i' ~ gene or other po'~ ' : ' can be faithfully and er~
delivered to the target cell. The result is either a transient ll ' :- r or a stable,, ~ D ~ mation or
the target cell.
SUMMARY OF THE INVENTION
This invention ~ ' a novel approach to gtene therapy and to :, 'etiof the immune system.
The invention relates to a . - that pr"~r,se~ the ability to localize r~ ' ~N~ ' -: ' to mucosal
tissues, neural tissues, and to other tissues or cells that display the GM1 receptor. The ~I ' '( : ' thus~5 delivered can be l' _ - genes or they can operate to modulate the immune
", of the animal. The
' ' may either increase or decrease the ' of a ' . immune response in the animal.
The invention is embodied in a c . for delivery of a pol~ ' : ' to mucosal cells, nervous cells,
and other 1,~. ' dl~ cells and tissues. The . - 5 . ioL5 a L- " _ protein having an affinity for
d~t~,.l of ~ b,dl~mucosaltissuesina ~ withanactive~vl~ ' :' Becausethe -' "_
20 protein also has an affinity for other cell types, the v . of the invention is also effective in delivering an
active ~ 1t to other selected cells and tissues as well.
A more particular - ' -' : of the invention has as the protein pr a member of the following
group of r~rns? ! " g proteins: CT, CTB, LT, bacterial fimbriae proteins, animal lectins, plant lectins, and lectin-like
proteins.
Z5 In a preferred - bc"I of the invention, the o _ protein is the cholera toxin of Vibrio
cholerae. This Sc " L C. ' leS the use of the ' ' . which consists of A and B subunits, or O
or G__ l thereof, and also o , ' the use of a particular fragment of the toxin, - _ only of the B
(binding) subunit. Also i ' ' is the use of only the A (active) subunit, involving both ~.__ : of the A
subunit (A1 and A2) or only one fragment thereof, as well as other I ~t ' '.~yhlg ADP li' yk~ activity.
Another: h " - of the invention has as its protein v Jm~ : an 1~' ' " capable of S~ ~;r~
110_., ~, mucosal cells. In a more particular L-' I of this aspect of the invention, the 19' ' "
It~LoL~ and binds to the GM1 receptor.
Further e-5~ " of the invention relate to the F"'~ ' ti ie c . :. In one bc " :, the
poi~, ' li ie is DNA. Where the p:'~, ' - is DNA, the DNA may be of genomic origin. All~llwli elt, the DNA
35 may be cDNA. A~'"- 'Iy, according to the invention, the DNA may be ~-- ' DNA. The invention
Culli . ~ - - all forms of DNA as the pcl~, ' li ie r . Jr
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W O 97/05267 PCT~US96/12041 4-
- This aspect of the invention has as a preferred - '~ DNA that contains a promoter region
3, , 'I~ linked to a coding region. The coding region may specify various protein domains. Another preferred
of the invention has DNA that encodes an antisense message.
In another ' ' of the invention, the F~ ' c , is RNA. Ar- P _'~, in a preferred
5 ! '~' t, the RNA is mRNA. In another preferred e ' ' t, the RNA is In an ~d'
' ~ ' t, the RNA is a ribozyme. The invention , ' all forms of RNA as the r ~ i.le c pr
The - , ~ may exist in several useful forms. All useful forms of the L , are t .'- '
in this ~ .~..; In one ~ ' Jd- t, the composition exists in the form of an atomized d-, ~ such as would
be used in delivery by ~ ' ' - ~ In a preferred r ' ' t, the composition exists in the form of a ~ s-~( y,
10 whether rectal or vaginal. In another preferred ' ~ ' t, the composition exists in the form of a cream, an
ointment, or a l~ llal patch.
The inventlQn ~!sQ ~ ...pl..!~ al! ways in which the nnucosa-binding pr~tein and the &ai~,.,u~ltolh~e may
form the r , In a preferred e ' ~ ' t, the . , exists in the form of a mixtute ~f ~a b- " v
protein and p~ wl~ The mixture may be stabilized by other ~ , If ' that exploit e' rt,u~ldli,, or
15 h~d..~' ' hll~aLliulls, or that diminish the ~rr~uli. of mucus as a barrier to delivery of the F '~ ' '
In another: ' -' I, the s. ! ~ " _ protein and the, ~ ' are !' ~ 11~ coupled to each other. In
an '' ' e...L~' of the i..~ the 1- ' _ protein and the r ~ t;d~ are , '
together.
Another aspect of the invention is a method for ~ ' ' - " immunity in an animal. The first step of the
ZO method is to - ' ~ ~ - to an animal in ' - a m~~osa l ' _ protein and a r ~ uti '~ encoding an
~ 19 The second step is to express the r ~ Iy ~ : - in the animal uhereby the animal g ales an immune
response to the product of the prl~..u..l~o '
In one preferred: ' " I of this aspect of the invention, the - ~ ' ' is an enhanced immune response
in the animal. The invention -l ~ , ' It.~ that the enhanced immune response may be mucosal, or systemic, or both
25 mucosal and systemic. An r''' ~' bc' of this aspect of the invention directs the enhanced immune
response to Cu-lllaC.,~,liuil.
In another preferred ~ bc' of the invention, the ' ' is a ' ~ ~ ' ' immune I r~- The
invention L ' ,' 1~5 that the ' ~ ~ ' ' immune response may be mucosal, or systemic, or both mucosal and
systemic. The invention is, ~r~laLly embodied in a ' ~ ~ ' ~~ immune response for ~ - to allergens, in
30 a manner ' v- to oral tolerance. In another preferred bor' I, the ' ~ ' ' immune response is for
; ' dLiull of organ and tissue 1, , ' A further preferred ' - ' L of the invention directs the :' ~ ~ '
immune response to the ~II a~ ..l of an .A~i disease.
According to the invention, the ' ' of the immune response may be done prior to exposure to a
pathogen or alleryen. The invention also ,' ~ the ' ' where it is done after exposure to the
35 pathogen or allergen. Therefore, all uses of the r ~ ~'- of the invention for the ' ' of the immune
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W 097/05267 PCTrUS96/12041
-5-
- response are embodied in the present I ~. . whether I ~ . ~ or F- . ~, and whether to immunize or
i7~ the animal so treated.
The . of the invention may be . ' ~ ~d in a number of ways. In one ~ ' ~ ' t, the mode
of - ' ~.. is nasal andlor, ' y. In a preferred b~ ' t,the mode of - ' ~ , is oral. Further
preferred ' ~ ' _' the pr of the invention rectally orvaginally. All~ ,the invention
is also embodied in a mode of _' ~ : i _ the ~ ~r of the invention by ' ' by _' tl i _ the
pr !, ' ' ' ~ dlly, and also by administering the composition transdermally.
Another aspect of the invention is the ~ . ~ r to deliver a p ~~ - ' to any GM1 animal
cell. The binding protein component of the s . ~ has an affinity for the GM1 receptor and because of the
10 affinity, delivers a r~ : to any cell ' ,' ~; " the GM1 receptor.
In one . bc ' : of this aspect of the invention, the bindiny protein is selected from the group ~
of GT, GTB, LT, LTB, bacterial fimbriae proteins, plant lectins, animal lectir.s, and lectimlike prs~eins.
In a preferred bc " :, the binding protein is either cholera holotoxin (with A and B subunits) or it is
a ' i.al;.~. of the toxin, having some fragment of the toxin, either alone or in ~ ' - with other protein
~I _
In an ' Ilali.., preferred: ' ~ ' t, the binding protein of the: . ~ is an ' ' "
According to the invention, GM1 delivery of a F~ ' li.le also . .': ~ purposes of gene therapy.
As such an ' -' :, the invention discloses a method for gene therapy in an animal by ~ to an animal
in ~ ' a GM1-binding protein and a r, : p~l~ ' ; ' and then i . _ the pr1~ ' liJe in cells
20 of the animal. By this method, the function of the r' H~ ~ o d~ confers on the animal a i' ~ . effect.
In a preferred e L " I of this aspect of the invention, the cells of the animal Ir ' '1~ express the
delivered rl ~ pL~ U~ u'''
An -' Ildli.~ preferred L-d exists when the cells of the animal are stably l,l.";.r. by the
r. p~
DETAILED DESG~lrllON OF THE INVENTION
Mucosal tissues are the route of entry of many ~- ' diseases. They are also the chief sites of
invasion and irritation by many allergens. This invention -' ~ases both of these medical problems. r: t,
the invention provides an approach to other Di~.alll diseases and ~ via a novel method of delivering
30 pGb~ ' lidd5 to certain cells for gene therapy.
The invention makes possible the ' ' liu,, of the mucosal and systemic immune systems of ~_. ' dle
animals by targeting E --' pN~ ..,lid~.5 to the cells involved in mucosal immunity. The pol~ ' li,les are
then ;"l~" ' ' within these cells and are ~ ased therein. The products of the r H~ t;d~5 can either enhance
or diminish the ~ n. !~ of the body's normal immune response to the epitope of choice, thus rendering the body
less reactive to certain allergens, or more effective at ~ certain F ' al, . according to the pal ~ of
. CA 02227871 1998-01-26
<
, . . .. .
the particular use of the invention. The ability to thus modulate the ,es~l",s~s of the immune system also relates
the invention to the Il~dtl"~"L and ~ iol1 of autoimmune diseases.
Rather than delivering only one dose of a particular pul~pe~ de allergen or imm~nogen, as would be the
case with a rQmrositinn COlla;aiilly ûf a mucosa-binding protein and a given polyp~,ulide, the present invention makes
5 possible a greater exposure of the target cells to a given allergen or ;~ ogen because of the fact that a single
polynucleotide can direct the eA~ r of many copies of its product. The ~tieLli.~,ness of the exposure of the
mucosal cells to the peptide product may also be improved because of the duration of the OA~ aaiun of the
pvl~,,u~ oIide within the cell, and because of the i"~, ~ " ' origin of the pGl~ product of the active
pOIylluLldrj lill~.
Because the mucosa-binding proteins of the invention will actually bind to any cells that display GM1 in
sufficient crn ll: . the invention further ~o ~ s a novel mode of pQI~l ' liil~ targeting and delivery fûr
gene therapy p~' ~~ Current modes of 1' ~, gene delivery are largely limited to viral capsids, cationic
lipids, . .,d live viruses, and ex vivo ll_ 'c ~ Most require injection of the material in questicn. However,
this invention makes possible the adr : dC of a Ih.,ru~ li.. gene or other ~ .liunal pGI'1llul~lu~,lille by means
15 of i"hd61- oral dosage, eye drops or creams, nose drops or creams, a ~pc-~;lu~i~3, enemas, douches, or llanade
patches, creams, or ~ ~ Auc.d 1~, the use of the present invention for gene therapy will have special
~r~. ~;.. against diseases of mucosal tissues and neural tissues, and against other diseases amenable to
by F ~ uLlUOi 1~ delivery to tissues the cells of which display 6M1 ,~c~l a.
SpeL;I 'Iy, the present invention discloses a c~-"~, for delivery of ~u"-,i ' pOI~llu~.leGlhle5 to~~ 20 various cells o~ L~al~.. The invention.. ,;;aea a binding protein andO a
r".~.i ~ ' pG~ 7lid~. The protein
l of the c , - has the property of r~e~,: ;..9 and binding to d~l~l. of many ~ ebldle cells.
lt may further have the property of protecting itself and other ' ' with which it is a~,oc~ d from rapid
d~O~dddliun within the body of the animal. And, being a,so ~lPd with a ~L.,Lliolldl pol~ ' -6dL, the binding
protein has the ;lllyullal~t property of delivering the pal~,,uLleuli,lO to target cells.
The present invention ~ I . ' le~ the use of any protein capable of ~ binding to or assoLiali.. 3
with any of the several neural or mucosal tissues, including dendritic tissues, tissues of the 9dall l~aIi"dl tract,
the IL~ ' _- _ tract, r ~ ~ r airways including lungs, nasal sinuses, and the L~ ~;.a of the eyes. As
. ' . the following proteins are all known to bind mucosal tissues: bacterial toxin ' binding subunits
including, at a minimum, the B subunit of cholera toxin, the B subunit of the E co/iheat-labile ~ rUtrjAill, Eordetella
30 pertussis toxin subunits S2, S3, S4 andlor S5, the B fragment of ~i, ' ib ia toxin and the l"~:"~I"ane binding subunits
of Shiga toxin or Shiga-like toxins.
Other m"ros? ~ ., subunits include the bacterial fimbriae proteins E. coli fimbria K88, K99, 987P, F41,
CFAII, CFAIII (CS1, CS2 andlor CS3), CFAllV (CS4, CS5 andlor CS6), P fimbriae, or the like. Other fimbriae include
Bor~ '1~ pertussis ~i'_.. . h...aOO' i . I/ibrio choierae toxin-cu.~,' t~ pilus (TCP), mannose-sensitive
35 k~ ~gJlni (MSHA), fucose-sensitive ' ~,O' i ~FSHA), and the like.
A~EN~
IPE~P
CA 02227871 1998-01-26
7- ~ '
Still other mucosa-binding " ~ include viral allachn~r"t proteins including influenza and Sendai virus
he",dyylui' - and animal lectins or lectin-like IIIL'e~ ' ~ including immunoglobulin mm~Aculps or ~rdylllrllla thereof,
calcium dPpP~ nt (C type) lectins, selectins, collectins or Helix pomati~ hemagglutinin. Plant lectins with mucosa-
binding subunits include concar,av '' A, wheat-germ agylul' ' phytohemagglutinin, abrin and ricin.
Other proteins that can aprL;~ recognize and bind to neural, mucosal, or other ~ell~bldlri cells are
cunl6lll~uldled in this invention. Speci~i,ul1y, any i" ir.~g'~t '' having an affinity for d;~li"tlN~ mucosal epitopes,
such as the GM1 receptor, is part of the mucosa-binding protein cornponPnt co~ ""~6led in the present invention.
Ad~'t'-- 1'~, lu '' l, chimeric, or Ullldl. ;ad er~y;"re~ed proteins po~sPs: Iy the property of binding to mucosal
cells may be used.
In a preferred e L~l lil l of the i"~,.",i' . the mucosa-binding protein is the cholera toxin of l/ibrio
cholerae. This L lil I conld,,,,uldl~s the use of the holotoxin, which consists of A and B subunits, and also
cout~,,l,uldles the use of a fragment of the toxin, Cu~ lg only of the B ~binding) subunit ,,~onulllrla either singly
or in Illullilllrla.
The invention is further embodied in the use of the A ~active) subunit of CT or its analogs as the protein
15 C ~ Ol,~l t of the cr,~l,os;li.~ This aspect of the invention c~r,l~""Jlal~s the use of only the A1 fragment, or only
the A2 ~ ', as the protein component of the present invention. Ad'"-- 15~, other enzymes, hGlll..,...,s,
COidLIula, or catalysts that, like A1, possess ADP-RbG~laliù,, activity or aid therein, also embody this aspect of the
invention. The invention further to~le"~ulales use of d~.i.dli.~s of the cholera toxin, whether natural or synthetic,
including all or part of either subunit.
20 ~ ~ Another ~nhG~ n~ the invention has as its protein Cv ~ ùl ~ ~ an i" o~lut_" capable of a~ut~ n!5
ecG~, ;..9 mucosal cells. In a more pd~ .ula, ~ ' '' l of this aspect of the invention, the il""~u..s_' ' ''
IrCG9~ ~ C and binds to the GM1 receptor.; 19' ' '' raised against other dr~rll ~s of mucosal cells are
also cu~r",,uld~ed in this i"~ni'
The binding and toxin ,u~uu~ S of the cholera toxin are well known in the art. Many other proteins with
-' 9~ binding plu,uLIlir.~ are also known. Still ûthers can be rllyilleelod. Likewise, ' o~lLt " - against GM1
or other specific epitopes of mucosal cells can be raised.
Further . bc '' la of the invention relate to the pol~,,uLleJ~iJe CG ~ onelll~ In one bt'' ~, the
poly"uLl,duGde is DNA. Where the pGI~lluLl~ûlidd iS DNA, the DNA may be of genomic origin. Genomic DNA can
be DNA with or without introns, and with or without promoter se9vru~e$~ enhancer ~y ,enre$, and other cis-acting
elements. Genomic DNA may also include DNA of genomic origin which has s~d ~eul ~ lly been Illald,Julàled by
recu,. 1,' ~ l DNA ie~bl ', -- Genomic DNA may be originally derived from animals, plants, fungi, protists, or
bacteria.
Alternatively, the DNA may be cDNA. For purposes of this invention, cDNA may be directly made from
mRNA via reverse l,dnsc,i,ulase, or it may be cDNA-like -' ' ~ where the DNA is not made from mRNA with
reverse l~dnsL~ laae~ but rather is '. '; ~d to remove introns and to add or remove other seu,~lrllce5 such that
the product of the I '. ' lions has the general chd~dLl~,ialiLs of cDNA.
lENl~E{) ~E~T
lPE~P
~ _ _ _ _ _
. CA 02227871 1998-01-26
~ 8- ' '', '
Ad''~'s--11y, acLo,u', to the invention, the DNA may be re~ ' ' t DNA. For purposes of this invention,
,~c L' l DNA may be any DNA that has been subject to l~ ' ' liùn by re~ - '' ~ l DNA ld,,h"i4.,J~. Such
i,~ , 3' may inciude, but are not limited to: random ~u~ r~e~;a sitc ~ led ",ul~9ane, N 3' or 5' RYn,..l.lea,e
trimming, linker addition, ligation with other DNA sequ~u~e,, Ill~ ldlion, d~ lhyldliulu polyad~"yidttu", insertion
S of a foreign fragment, and removal of an e-~c~, fragment.
The DNA may also be random ~Idylll~nls of DNA. For random ~Idylll~llldliui1, the initial DNA may be a
selected amplified sample of DNA, or it may be a random sample of genomic DNA.
ACCGrd;"YI~, the pûly,,ul.lLûlidd component of the invention is not limited only to s~ n~es that encode
protein products. Any fl -:iuual DNA sequence is r '~ ,' led, without reference to the length of the molecule
10 in base pairs, and without reference to the physical structure of the molecule. Therefore, whether the DNA is in
plasmid form, either ~ " ' or open circular, collcdldlll~li." linear, single-stranded, or double-stranded, the
invention cGIll~lllpldla~ any ~u"..t' ' DNA sequence as the pol~l,uLl~u~id~ CG r~ '
This aspect of the invention has as a preferred . Scl' : DNA that contains a promoter region and a
coding region. Where there is a promoter region, pl. lela of any origin are culll~ JldlLd.
Coding regions may specify a palli.,~ldl p- 5~ . Such coding regions may include introns or may be
without introns. In addition, other kinds of coding regions may be used. Examples of other kinds of coding regions
are , u that encode an antisense message or , r S that co"sli~ targets for DNA binding
proteins or other agents of i.-l~- 1L ' ~ i~leld~liv~
When the coding region does specify a ~ ,_,,lide, the invention . I~ s all forms of coding regions,
20 both naturally occurring and l-~ bi l. The s, ~ss may be derived from random or sitc ~ Lled
or they may be non-mutant ~e~ \.eC They may also specify various protein ~ Jcll..e;. and domains, such as
helices, sheets, signal peptides, transit peptides, targeting domains, retention domains, I~y~LLp~~' domains,
h~d~.,'"' domains, fingers, ~ippers, loops, coils, active sites, binding sites, processing sites, cleavage sites,
o''~, iLali~n moieties, and the like. They may also specify random ~, of known or predicted protein
25 ~ ~e s
This aspect of the invention is further embodied in DNA derived from a virus. Such DNA may be directly
purified from a ONA virus or may be purified from a viral host, whether r ' yLJIi~. or plû~al~uliL In addition, the
viral DNA may be ~1llDl~aiLed in vitro. For purposes of this invention, viral DNA includes any DNA molecule of a
virus or derived from a viral RNA or DNA selluProP whether the sequence is complete or only a portion or fragment
30 of the sequence is le~,.e.~ d.
Another preferred; bc " I of the invention has DNA that encodes an antisense message. For purposes
of this invention, an antisense message is any pnl~ ul;d~, sequence capable of ~ , ' laril~ assor.:-li. g with
other p~l~nl~LleGlid., s. l ~res that would Olhd~ e be ll~n,ht~d, or would assist in lldn~ldtion. This definition
Lr~r~ paC5as p Ntl.ULl~otilJds that can form an antisense hybrid with, for example, MRNA, tRNA, rRNA, or other RNA
35 -' - 'os, regions of RNA '~ ' . or other active pcl~l.uJ,col:dus. The essential nature of such anli~nse
AMEN~E~ IE~T
IPEh~P
CA 02227871 1998-01-26
Y . '..; .-
",Pss~ges is that they are se~n~",es capable of binding to other polynucleotides and either eliminating or all~uiJdlillg
their ~lai1sldliun or other r~lraaiùn or activity. DNA encoding an antisense RNA may aiso be used.
In another Pmhodilnrl l of the invention, the poly,,ul~leulide cuu~,uonrlll is RNA. All ~ullLliunal forms of RNA
are corllrllluldlrd in the invention, regardless of origin, structure, ~uu~ alions, or method of ~JuR~i~.ation.
In a preferred elllb I - 1, the RNA is MRNA. For purposes of this invention, MRNA is any RNA lldlls6lrd
or capable of being lrdnJdled to create a pol1l,e,uli-lr product. Therefore, MRNA can be purified from cells, either
euhd,~uli-, or prokaryotic, or it can be S~lllh6a;~l3d in vitro. The invention co"le",,ula~es MRNA 'e ' - with and
without l,lGd;~iLaliuns affecting el~ ;rllLy of ilcms;diiull, such aa, fûr exampl~i, po',~de;;y!d!iun at thP 3' Rnd or a
5' m'G cap. In another preferred E" ho ' , the RNA is antisense RNA. For purposes of this invention,
antisense RNA is any RNA sequence capable of cv, ' La,;l~ binding with other pûN~IluLl~duliJE se.~u~es that
would ull., .~ iad be lldllalalrd~ or would assist in ll '. This definition e ~ a~ses p~lyllu~.lruli~lda that can
form an antisense hybrid with, for example, mRNA, tRNA, rRNA, or other RNA I -' ' regions of RNA ~' ' s,
or other active polyl-u..l~Glidda.
This aspect of the invention is further embodied in RNA derived from a virus. Such RNA may be
15 directly purified from an RNA virus or may be purified from a viral host, whether eukaryotic or prokaryotic. In
addition, the viral RNA may be synthesized in vitro. For purposes of this invention, viral RNA includes any RNA
molecule of a virus or derived from a viral RNA or DNA:, ~e whether the sequence is complete or only a
portion or fragment of the sequence is Ir.,ulrs~lllOd.
In an 3d~ _ IJc' 1, the RNA is a ribozyme. For purposes of this invention, a ribo~yme is any RNA
20 mo~cule with a catalyt!~ activity, whether aull,calal~ . or hELelu~.dlLl~lil,. The use of any ribozyme as the
pol~"u-.leui '- cc...,o L of the c ~,nGsili, of the invention is cGIll~ Jldled herein.
Other forms and cG..~u..,.dLiu~a of RNA are also part of this ;,.~.,..: As PY- 1f the RNA may be
rRNA, tRNA, hnRNA, single-stranded, double stranded, self . - ' d, looped, or cu..cdtdlllrlil..
Ar'' - ~r 'I~, the r N~lluLleulild C_ r I of the invention is embodied in any D --~liunal poly..u~duûi ' ~
25 that are not listed above, whether existing in nature or synthetic. Examples of such pOl~llul.lrulidds cGIllell.~Jldll,d
by the invention are: linkers, genes, ~JIlJ,.lulrl~, introns, enhancer seu, r~ces~ silencer se.~ es, t,_ ,o ''
elements, RNAIDNA hybrids, RNAIDNA chimeras, DNA ~ CGn~ E~ ..u..leùtide analogs, RNA corii li
llu"l~uli.ld analogs, hr~ ., ~ mixtures of different DNA Se~lur ~es, helelugrn~ùlJa mixtures of different RNA
,e~ "~es, and hrlril-3 -o mixtures of both DNA and RNA seg,~
The , . - may exist in several useful forms. All useful forms of the co, ~I G. Iiun are cû"lr.,l.~laled
in this invention. In one L~r' l, the c~ uoc;l; n exists in the form of an atomized di~,r,.s;u.l for use in delivery
by i..l.ahliun. The atomized d;~rla;on may be a solution of the c- - of the invention, or it may be a
- -r ' Cuula;ll;uEI the co,~uos;liun. Typical carriers for atomized or d~:lus~ rd di~urra;ùllas include MiglyolB',
Frigen~ l1111211131114), and buffered saline. The delivery of the cu ~pcs;l;orl of the invention via i"l,ahl;un has
35 the effect of rapid d;~ue~a;ù~ to a large area of mucosal tissues, as well as absu,pliun by the blood for l,bLuldliun
of the c , of the invention to other GM1 cells of the body.
AMEN~D SHEET
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In a preferred bcl l, the c , ls n exists in the form of a, I ~o~;lo-y, whether rectal or vaginal.
Typical carriers for t~ lulalion of the inactive portion of a s~lppository include poly~ le,.e glycol, glycerine, cocoa
butter, I ~ c~.~ H, and Witepsol'~' H. Other ~ 9~PGS;~ fulll~aldliùlls suitable for delivery of the cG,..I)o~,liun of the
invention may also be used. ~elivery of the CO I GS I; II of the invention via suppository has the effect of contacting
a mucosal surface with the co l;~G~;Ii Ol for release to proximal mucosal tissues. Distal mucosal tissues may also
receive the cu a,r~i(io,~ of the invention by diffusion.
A~ 'Iy, the invention cuui , ' ~ the co ~,~c~i~ivr~ existing in the form of a liquid. The liquid may
be for oral dosage, or for û~Jhi' ~' or nasal dosage as drops or for use as an enema or douche. When the
composition of the invention exists in the form of a liquid, the liquid may be either a solution or a c ~pPu~ rl of the
. - n~ There is a variety of suitable tullllulal;Ons for the solution or "~pP c ". ' r ~ on the intended
use thereof.
Delivery of the c~ ~O~IiO,I of the invention in liquid form via oral dosage has the aim of exposing the
mucosa of the ga~ ali"al tract to the co- ~ . s;li-~ - A suitable dose, stabilized to resist the pH extremes of the
stomach, would deliver the c ~I G. Iiu" to all parts of the gaallL l~li"al tract, especially the upper portions thereof.
The invention c ,' lts all means of: '" the crl pc in a liquid oral dosage such that the effective
delivery of the c , ~ could be evenly ": Suled along the ga ,ll, le;~lillàl tract. For example, a coated or
c~ release material can be used to release the ~ ~p~ n only after it traverses the stomach. For oral
adm;.,;~.l,ali~n in liquid form, the - pr could be thus coated in fine particles and be ~ lod as a
Delivery ~e he co ~p~:li l of the invention ~ liquid form via ophthalmic drops wou!d be with the aim of
exposing the mucosa of the eyes and ~,o~ Pd tissues to the c -s A typical liquid carrier for eye drops
is buffered saline ~ " Methocel~ 65 HG 4000.
Delivery of the o ,- - of the invention in liquid form via nasal drops would be with the aim of
exposing the mucosa of the nose and sinuses and assG~,;àl~d tissues to the c , Liquid carriers for nasal
drops are typically various forms of buffered saline.
The invention is further embodied in the c~ ,, in the form of a cream for G~Jb~i' ' ', nasal, vaginal,
or rectal admi";..ll ~i The use of the c ,- of the invention in the form of a cream applied to a given
mucosal tissue has the aim of ti"g the C~ ?r~ on with the mucosal tissue for a auttiL;~"l time to allow the
effective delivery of the c - to the tissue. The cream may also have ~ of ss' ' " ~, mucus or
30 olh.,.~ ;je ~ , the effect of mucus as a barrier to delivery of the c r~ Ad~litirn~MI,ucol~li.. ayents
such as DNase solutions may also be added. Typical creams and . use as carriers paraffin oil, Pt:llUIdlUIII~
glycerol, pGl~lh~l~ne glycol, ~I~~rt~ glycol and Methocel~b
The invention is also embodied in forms of the c~ ""iàtl: for topical ap~lic, I r, or l,a"sde""al
delivery to dendritic and other cells. Cu..l~ J6led forms include ua"ad~ll"al patches, creams, and Gi..llll~lll~.
35 Tlàllsdellllal delivery has the aim of releasing the C.~ ,c~;lh"~ either rapidly or gradually over time, to cells proximal
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~ . . ~ ... .. ..
to dermal tissues. Such cells include dendrites and other nervous tissue, blood cells, and smooth and striated muscle
cells.
In another emho~;r.A,ent of the invention, the composition exists as a solid tablet or in a capsule form, for
oral dosage. The use of the c~.."i~o, liu~ in this form has the effect of delivering the cQmro~ition to the
5 gas~,ui"lesli"al tract for exposure of the intestinal mucosa to the cu,,,po~;liun. AccorJ ~ly, the tablet or capsule
may consist of a material effective in plul6Llill~ the c~ l,o,;lion from pH extremes of the stomach, or effective in
releasing the cormrositinn over time, to optimize the delivery of the co,,~l~Gs;liun to the intestinal mucosa. The enteric
coating for acid-resistant tablets and capsules typically includes cellulose acetate phtalate, u,u,u~l~,,e glycol, and
sorbitan mon~ t~
The invention also coul6"~l-ldles all ways in which the ml~coC? ' ' ~, protein and the pol~lluLl~uliJe may
form the CO l~ j,;l;'Q In a preferred emhodi~nt~ the pol~,.uLl~uli.le and the protein are mixed, without the
i"l~"Oùnal ~u~ dliun of covalent bonds between the polyllu.,l~oliJd and the protein. In this Pmhod 1, other
illyl~di~nls may be included in the mixture to enhance and stabilize the asso.,;dliu" between the protein and the
poly--ucleo~iJc. The proper mixing ratios of the protein r , and the pol~,.uLl~otide cu.",uone"l can easily be
15 L ,- i -'Iy d~ ' d~r ~ ~ on the desired effects of the mixture.
The present ways of stably but noncuv ' lly joining a binding protein and a p~'~..ucl~ulide together include
the polyca~; polyly~;..e method and the cationic liposome method. In the p~lyl~i;.. method the binding protein
is first rl :, 'Iy coupled to pol~ ;..a. The pGI~ ~ pol~'~ ~ coupled with the binding protein is then mixed
with the polyl,ucl~olide. The el.,.,lllJsldli.. ;..lelaclion~ between the pQ1yly~;..e and the nucleic acid create a stable
20 cnmp~)sitp~n that ~oes no~~easily di ~ t and is ~herefore suitable fo~Odelivery to the target cells. Wu, G.Y.,
Wilson, J.M., Shalaby, F., Cru~ a~ M., Shafritz, D.A., and Wu, C.H. (1991) J. Biol. Chem. 266:14338-14342; Chen,
J., Gamou, S., Takayanagi, A., Shimizu, N. (1994) FEBS Lett. 338:167-169.
Another example of the ~c h. ~,~ is the use of positively charged lipids to form li~ -s capable of
_I~_IlU~Idi- -'Iy illl~ld~.li..~ with P~1~IIULI~OI;d~;~ The pcl~llu~ ui ~ is mixed with the lipids and 1 s are
25 formed. A Li~"..Llional L pc I of the liposome is then chemically coupled to a ' i.àli~l:d form of the binding
protein of the i..._.,i The , - is thus capable of delivering the pGI~ ' liJe to the target cells without
the for",aliù.. of any covalent bonds between the polylluLl~oliJ~c and the protein. Mizuno, M., Yoshida, J., Sugita,
K., Inoue, I., Seo, H., Hayashi, Y., Kn~ a T., and Yagi; K. (1990) Cancer Res. 50:7826-7829.
In another h-'- 1, the mucosa-binding protein and the p~ IiJ~ are ' ~ coupled to each
30 other. Various linkages are co,ll~..l,uldl~l. As ,' the linkage may be either at the carboxy terminus of the
protein, the amino terminus of the protein, or at an R group of an amino acid residue within the protein. The
pol~"e 1 .,lil may be linked at the 5' end, the 3' end, or with some ~al~"uLl~otide cGIlro~ alions, the linkage may
be internal to the polyll..cleul;dc sPqnPnrP
In another preferred h~ " l of the i....: . the mucosa ~ " ~, protein and the polynucleotide are
35 A ~p~ d together. The ~ S ~l~liOIl means may play the dual role of I I ;, the assoL;aliun between the
r- , - of the c~ -\I.u,;~ while also ~ulul~.li,,9 the r - from stomach acid, digestive enzymes, t Al..eS l;~
A~FN~ gHE~T
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. CA 02227871 1998-01-26
mucus, or other obstacles to optimal delivery to the preferred mucosal tissue. The great variety of possible
Pn.~;s~ means is well known in the art. The invention cG~ llluldl~s the use of any Pnc~ J means
suitable for a pd,licula, o,,' lioll.
Another aspect of the invention is a method for modulating immunity in an animal. For purposes of this
5 ;"~, I"GJ~6lilly immunity includes either e~ or ' ' ~ an immune response, or it may also include
effecting both ~n~ ul and ' ~ , as ~ s~ed below. The first step of the method is to
a~' ~ to an animal in r ' lion a mucosa-binding protein and a poly"uLleuliJe encoding an; ~,oge-- The
second step is to express the pol~ lida in the animal, whereby the animal gene,dlGs an immune response to
the product of the pol~.J~ ulille.
In one preferred embodiment of this aspect of the invention, the Il10[1UIdliUll is an enhanced immune response
in the animal. r1e' ' ~ for 6 ' - l may be lhaldl,l~ Gd by a more rapid immune response than would
ollldl- 6e occur. It may also be ctldldt.lG(i~ed by a higher titer of ~9' Sc' after exposure to the antigen.
Adfitinn ~' ,,ha~d..leliali..a may include an increased ~Gaisld"te to a pdibe9 , a shorter duration of infection by a
pathogen than would ul' ~ ;ae occur, an increased reactivity to related palhrJgeus or antigens, or increased
15 illrldlllllldtùry response to the antigen. The invention C~--u,~ S that the enhanced immune response may be
mucosal or 5y or both mucosal and systemic. ~ -- ! ' O proteins such as CT are known to be effective
in delivering pcl~ .l;J~,a for yellGI - an immune ,l re Use of these proteins to deliver pol~,,u,,lLuli'~
encoding antigens is similarly effective.
In addition to the ' -l a of enhanced immune lesuoi,aes listed above, a mucosal --' ' immune
2D ~ response may be ~hdldl,leli~d by an i~reased prl,JuLliuh of n~l~dl;L;Il9 ~;LQ ' including slgA. It may be further
clldrdl,lGIiLGd by i"wGa,_d, h: of ~ 19~ ' ' IgE, IgG and by enhanced cell-mediated immunity.
In addition to the ' ~ra of enhanced immune response listed above, a systemic e~ Fd immune
response may be ,.hdl~LlL.i~Gd by an increased l,.uJu..G of mGulldli~ill9 ant;t,~'!~ including lr. ~' ~ ' IgG.
It may be further clldldLI~.iLLd by increased ~ Jr of og'ot ' IgA and by enhanced c_llmo~i~ted
immunity.
An ~'' ' . ' ' l of this aspect of the invention directs the enhanced immune response to
CG~Ill L ,:- An acquired specific mucosal immunity to delG" ~ unique to sperm cells is c" u~ led. Where
such an immunity is ~, dlGd~ the animal so ~ !d would react to clear mucosal tissues of sperm cells, thereby
dGL,.P ~ the number of active sperm available to fertilize an egg of the animal.In another preferred embodiment of the invention, the IlluJuld~iull is a f- ' ld immune response.
'1e-' ' - for a ~" ~ ~ hnd immune response may be cha,dcl~ ed by a less rapid immune response than would
oll,e,~ ;ae occur. It may also include a lower titer of imn~lnog~o~ ~ after exposure to the antigen. Additional,
cll~ltll,l~liali-.a may include an increased tolerance of an allergen or related pdlhoy~l\a or antigens, " ' ed or
F'' ~ l~-l allergy a~ lla, or down-l~yuldLi~... of i"~6l"",alu,y cytokines. The invention colll~lll,uldleâ that the
35 ' ~ ' ' immune response may be mucosal, or systemic, or both mucosal and systemic.
A~EN~ ~HE{~T
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. ... - ~ ~,;v,,
The inYention is ~l~r~,dbly embodied in a d ' ed immune response for das~ i"3tion to allergens. The
invention cun~ Jldl~s varying degrees of de~n~ dlion.
One example of ~ai~ lion to allergens is the well known rh~ ~ of oral tolerance. In classic oral
tolerance, an animal that is exposed to relatively large quanlili~s of a pa~ lil.uldl s ~l ,Ih~CP for example, an alleryen,
5 over time becomes less c'~5 ~ sensitive to the allergen. The oral tolerance thus d~.aloyed can manifest
itself both in the mucosal immune system and in the systemic immune system.
The dosage required to develop oral tolerance can be ~;y"i~i,,d"ll~ reduced using mucosa-binding proteins
coupled to the allergen. In eAu~,i,,,ellls delivering human gamma globulin (HGG) to mice either via simple oral dosage
~feeding) or with a v ~ protein ~CTB) linked to the antigens, the amount of antigen required to achieve
the same level of tolerance to delayed-type hy,url~dllsil;.ily (DTH) (late phase) was d~ oAillldlGly 200 to 625 fold
greater with simple feeding than with coupling to CTB. Eul Ihellllùlr~ the delivery by simple feeding was i"~r~ e
in diminishing the early phase of the DTH response, while the delivery of relatively small doses via coupling to a
mllrosa' " ~ protein was fully effective. Sun, J.-B., Holmgren, J., and Czerkinsky, C. (1994) Proc. Nat. Acad.
Sci. USA 91:10795-10799.
The present invention likewise exploits the capacity of the mucosal immune system to ~ nudlr the body's
immune response to certain allergens or other ;~ -.o~ ..c, but not by delivery of protein allergens or ;~ --- ,9. "s
lhl:".;..,h,.,~. Rather, this invention delivers active pol~,,ucl~ûliJes to the mucosa in order that the products or the
activities of the p~1y, ~' :iJes may play a role in the diminution of the body's immune ru, It is important
to note that the p~l~ ' liJes Ihdll.~ .,;. are not the 7 . antigens, allergens, or haptens, but instead it
20 is the pol~Jd,ui ' products, or the other activities of the p&l~uul,k,~.liJ.,s, that ullillld~ly effect the de~ired
d'- ;- of the immune response.
Because the pdllil,U6l protein CUI~ O~ --l of the C~J ,p~ :~; has a great effect on whether the immune
response is ,~ uJ~ ' n~d toward ~ ---l or ' ~ i . the invention c~ u~ 'r~ Cu~ ~p -:0~ capable of either
kind of u~e~ dl; n, as well as -' ' lion of either type of immunity, whether mucosal or systemic.
In another preferred '~' :, the f ' ' immune response is for i ' of organ and tissue
Il ,' E~ nonself signals of the MHC I class of proteins are capable of ~ an immune
l--r~ . and their genes are therefore ca"d;ddl~ for use in the C~Ul.r5~ liv~ of the invention in order to eliminate
or at least di le the immune response assoc;alrd with l~coJ, :~d nonself epitopes on lldn~Jldlll~d organs and
tissues.
As a example, delivery of a gene encoding an MHC I protein to the mucosa via & ' with a mucosa-
binding protein results in local rA~I~aah~n of the gene in mucosal cells. The gene product, rL ~ ~ nonself
epitopes, would normally stimulate a ~;y"iri"d"l immune response. Instead, because of the nature of its l:A~
in mucosal cells, it effects a ' ' ' s~"~ ;ly to the epitopes, which " ' ' al~ il;.;ty iS manifest in both
the systemic and mucosal immune systems. Shortly th~l~drld" a lldll~pldlll that displays the same epitopes is placed
in the animal, and the immune response directed against the ll~ lad tissue is much weaker than it would
A~EN~ St~EET
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ulll~ll ;ae have been. ACL...-IjUYIY, the chances of rejection are deL.~ased, as is the need for -' IdOull of
immune-au~J~(e~a;on drugs to the tl.ul~uldlll recipient.
A further preferred L-d I of the invention directs the !'- ~ ~ ' ed immune response to the ll~dLlll~
of an ~. - disease. F " - nonself epitopes are not the only d~l~- Is Irco~: rd and attacked by
5 the immune system. Some ' ~" of the immune system result in a lldJl~rrJ9~ of self as nonself,
a p, ~ ' by a mistaken immune reaction against the animal's own cells and tissues. This ph~.o ~r~")~ is the
basis for dUi ~ diseases such as, for example, systemic lupus erylll~llldlJ~ type I diabetes, ~hdu~dl~.;.l
arthritis, and multiple sclerosis. Where such a condition exists, ~e,~ lion of the immune system to the
c~ rd J~. ~ ~ of self cells ' l~ or ' dlt~ the cor~;tio~ Acco,d;.,~l~, the invention
10 c L~ Jldl~s the use of the -t pc ~ - of the invention to treat or cure dUi ~ InP diseases.
Also vv ,' l~d in the present imention is the ~: against certain epitopes of a disease
pathogen together with a sin,~'~ d~ i".li.~" to other epitopes of the pat~ogen The effect of such a
simultaneous up- and down- I O ' of the immune system is to focus the effective immune defenses on the
pathogen so as to eventually control or eliminate it, while reducing l.y~ld~ l tissue" damage due to I, ~ d ~;.
15 collateral ;..r . In cases where different epitopes stimulate the cell-mediated immune response on-one hand
and the injurious " : y response on the other, the disease can best be treated by s~ c I~Gdu6i
of the immune response toward greater se~;l;.;.y to one epitope and much less n .~ ~.-,.-_5S to the other.
According to the ..: . the d~!i.: of the immune response may be prior to exposure to a pathogen
or allergen. Examples of 9" ' of the invention using ~ A~G3~I-Dd~ include v c; .
20 ~e~rn~ i . and p~b~u~JIiv~ ~u~c -1~
Where a pathogen is known, and v. ~ ~ :- against the pathogen is desired, a gene encoding an epitope
of the pathogen is delivered as part of the - pr of the ~..i The gene is i..l~.. ' d and eA~ ..sed within
mucosal cells, and an 6 ' ~ i immunity of both the mucosal and the systemic immune systems results. Suh,~
exposure to the pathogen is met at the point of contact by a strong and specific mucosal immunity as well as
25 systemic immunity. The ~r~GIi.. of the pathogen as a disease causing agent is thus reduced or diminished.
The invention also c , ' the modulation where it occurs after exposure to the pathogen or allergen.
Examples of 9, r~- - of the invention using pQ~ ' ' - include pv -- ~ for chronic
~ ',: : to create an ~ reaction to cancer cells, d~ dll........................... l for allergies, and
to epitopes attacked in : disease.
Where an . - ~ disease has been ~; g,-~se~ and its basis is _ ' ~c-d, a p~ .. ucl~,liJe encoding
the epitope being ~LG~ ~d as nonself by the immune system is c ,' ' or oll.~.~ ;ae combined with CTB.
The c . - is fed to the animal in an ~,, ., iale dose, which results in !'- ~ ~ ' ' or " L~d mucosal and
systemic immunity against the epitope of the dUi ~ disease.
ALC~ , all uses of the of the invention for the od ' i lr. of the immune response are
35 embodied in the present invention, whether iJIL~A~u~lJlt or pGsl~A~JGaul~ and whether to immunize or de~ r the
animal so treated.
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CA 02227871 1998-01-26
The cuu~ ion of the invention may be e ' dd in a number of ways. In one ~ od~ , the mode
of ad ~.dliUII is nasal. ~r~ IdliOII of the cG,~nos;~i n of the invention nasally has the aim of exposing the
mucosa of the nose and sinuses and as~uLidl~d tissues to the co~rosition All~llldli.Lly, the invention is also
embodied in a mode of -' : i"g the c Inos;l;m of the invention by illbdldliOn. A~ dLion of the
5 c~ pû,;li, of the invention via i"hdldlio" has the effect of rapid .~i~p~,~;ùn to a large area of mucosal tissues, as
well as abso"uliùn by the blood for cb~.uldliuil of the cv, ~I~Gc;li~n of the invention to other GM1 cells of the animal.
The invention is further embodied by ~' ~: b~y the c~ n ophi' ' -11y. ~ ~ ;ldliùll of the c~",uG~;~ion
of the invention ~"~hih~ has the aim of exposing the mucosa of the eyes and associdled tissues to the
C - - r ~ ~
In a preferred e~ - --I, the mode of - ' .. dliun is oral. Al' ~ lldliuo of the co",l\o~ li n of the
invention orally has the effect of exposing the mucosa of the gasl~ li"al tract to the CG~ JGs;liu". A suitable
dose delivers the Cv-~l-G~ o to all parts of the gd~l-l Idslb~al tract. The invention cGnl~lllulal~s all means of oral
delivery, whether in a liquid oral dosage, as a solid tablet, or in a capsule form.
Further preferred eulllGl~ l ~- the c l~pu,;lioll of the invention rectally or vaginally.
15 ~I' ~ '.dliull of the co ~",5;1; ~ of the invention rectally or vaginally has the effect of cvul. Ii- y a mucosal surface
with the cr-r~ for release to proximal mucosal tissues. Distal mucosal tissues may also receive the
c ,: of the invention by diffusion.
Another aspect of the invention is the ~L , tllllLi _ to deliver a pr,l~,,ucl~uli le to any GM1 animal
cell. Where the binding protein L , of the composition has an affinity for the GM1 receptor, the r , - ' -
20 could be delivered - ~ any cell d, ' ~;"v the GM1 recs~to~. .
GM1 v ' - ' is found in the plasma l"~"iL. - of cells 0ll uu,vllGul the body. However, the CollC~ d;
of GM1 in the plasma ",~",br of different cell types varies ~;y"i~iLd~ . Only in cell types with a certain critical
level of GM1 tonc~"l,dl;on does GM1 actually act as a receptor for binding CTB. Different binding proteins interact
with GM1 or dyyl~ydle~ of GM1 in different ways. This fact allows a person 1-- - _ the invention to design a
25 gene therapy ~ r capable of limited targeting of a t' ,.~,.,..IjL gene to mucosal cells, or more general
targeting to other cells Fcss~ , a certain concL..Irdt of GM1 in their ! b~. 9~ This mode of delivering
h~lù~ iL genes therefore allows a degree of precision in selecting target cells not ,J~b-;.JU~ available.
The present invention cGrll~llluldtds the use of any protein capable of ~JeLiri 'l~ binding to or dSsuL;dlillg
with GM1 cells. For purposes of the invention, a GM1 cell is a cell of a ~ Lldl~ animal having g_ _' ' GM1
30 in its plasma ",~",L.dne. Many of the proteins already listed before as mucos o ! ' _ proteins are either known
or believed to bind mucosa via the GM1 receptor, and are therefore also suitable for use in this aspect of the
..C Likewise, all of the kinds of p.~l~llucl~uli '~ d; ~ d above are also a~.~".r id~d for and cor,l~ p'-lPd
in this aspect of the invention.
The delivery of the c~ ~u~ of the invention is not limited to mucosal cells alone. Many other cells
35 display GM1 in varying cGr,ce,,l~dliu,,~. For example, neural cells are known to have high conL~"I,dliùns of GM1.
Other cells whose surfaces have lower collL~ulldli~.as of GM1 may be primed for delivery of the composition of the
~I~EN~ ~EET
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invention by ,u,u;,,Lubaliùll with GM1 followed by a si '" period, then finally followed by dosage of the
cu""3Gs;liun of the invention. Cells with different Illcu,lJ~dne cGIllpollcllls will have varying degrees of affinity for
and rates of uptake of GM1. Acco, " ~, the invention can be practiced in such a way as to regulate the time and
other con~ c of illbubdliv.., together with the conLc,ll~dliùn of the ~.ogr"au. GM1 and the time of : bi" lion,
5 to modulate the crFcLli.~..css of the uptake of GM1 by the target cells and thereby modulate the ultimate dosage
of the cu--~poc;'i- n delivered. Thus, any cell type can potL 'Iy be aclcLL..,ly ~,cl,edled to enhance the erri..;c"~.
of delivery of the 'r-- of the invention.
Likewise, anti GM1 antibodies can be used to block GM1 receptors and dllcnudlc the affinity of high GM1
,...,..~brà"es for the & , ~ of the invention. The invention dllli..;~udlca the use of both forms of ulcllcdllllclll.
10 either with antibodies against GM1 or with GM1 itself, either singly or in s ' ~ :- . to achieve the desired result.
As an example of the use of both forms of plcllcdllllclll~ a tissue co~ ,g of two major ~ ù~(n l'liùn5
of ceils is selected for llcaL.. .~t with the low-GM1 cells being the most preferred target for delivery of the
c ~rG~ Iim of the ~ .. First the entire tissue is treated with a solution CGlli ' ~ _ anti-GM1 IgGs at an
15 au~ulopliàlc concc,,l~di The IgGs occupy most or nearly all of the: ooes ' ' GM1 in the ' a"es of the high-
GM1 cells. After lavage to remove the excess antibody, GM1 is added to the tissue. Following the diffusion
gradient, GM1 will insert into the low GM1 membranes more than into the high GM 1 membranes, and the GM1 thus
diffused will be largely free from antibody blocking. After a second lavage to remove non i I-cd GM1, the tissue
is ready for dosage of the s pr- of the ;..~. . and specific delivery of the active pol~ ,,lcuGJ-c is effected
2D to the cells tha~ ~ere or;~ally lowest in.GM10
The gene therapy aspect of the invention discloses a method for gene therapy in an animal by r ' ~ ~ j~
to an animal in - ' a GM1-binding protein and a r~"..,lio,lal pcl~,..,,lcoliJc and then cAlJI'caa;ll_ the
pGI~Iluclcul;J~ in cells of the animal. By this method, the function of the pGl~,...,,l,,~GJc confers on the animal a
Ih~P~ ;L effect.
In a preferred ~ bc" of this aspect of the invention, the cells of the animal llal,a;c"ll~ express the
delivered t~ ool~ 'I li'u. Transient -ca~ ~ is Lhdldl~lcli~'cil by an onset of eA,uldsa;un cl,. 11~
followed by a cessation of e..,u,- The duration of c~,u,, ~ is a function of the stability of the active
pol~ ' . ' in the cell. Factors affecting the stability of the pol~,....,lcuGJe include: the nature and structure of
the pol~lublcoi ' . the subcellular location of the po~rllu~.bculiilc, the d~ , of l ' ' proteins or Iytic
30 enzymes in the local area of the ~el~..JLI~.~li-' and other physical pd,d",clc,s of the cell such as lc~ dlUIc~ pH,
and conc~i..lld;- of certain ions.
For many , ' : s, transient -c.~,u,G..a;ù,, is preferred because the product of the delivered gene or the
activity of the F~ ' would be d~lulcl- in the long term. Therefore, in such cases, selection of a
pr1~,..J.,lculida more likely to be lldlla;cllll~, and not stably, -c~u,cased, will be dpp,uplidl-c. The cl,a,aLIc,iali,,a of
~5 pol~ oti~s that render them more or less stable in a host cell are known in the art, and a person of ordinary
skill could select an 3,, , ialc pul~l,u-,lculiJ-c for the pdlli~.ulal practice of the invention.
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An all~,l,dl;.~ preferred embodiment exists when the cells of the animal are stably transfDrmed by the
lu"cliunal polyll,JcldGtill~ Stable llar,~urll,dlion is characterized by the actual hll~yldliull of a polynucleotide into
the genome of the host cell, and sllhse~luent ~x~s~;ùn of the genes or other activity of the poly"ucle~liJe
Ihl._o':~_l the life of the cell.
Certain uses of the present invention will neces~;ldl~ or benefit from stable tld~sroll''dliun of the cells by
the pGl~l,u~,le.,lide. For example, where the therapy involved is the rrp~ l of a missing or nGIlRlllLliundl gene,
stable llauaiulllldliun of the cell would llo~ dll~ cure the defect, where transient ~A~ aaiUa of the pol1llucleulidd
would u~cesaildle CG~li' ' O t,~al,ll~"ls.
The ci-ldldl,l~lialil.s of polynucleotides that may alone or in c ' liun make the pGI~-.ucleulide more likely
to stably integrate into the genome of the host cell are known in the art. A skilled artisan could select the desired
,hdldLl~lialiLs for the polynucleotide to be delivered so as to maximize the likelihood of stable illl~yldliol1 and
Il d~ Illdliùl) uf the host cell.
EXAMPLE 1. Formin~ the CUIII~.J;~
Cholera toxin B subunit protein (CTB) is coupled to poly L-lysine as described ~ iu~lal~ . Jung,G.~ Kohnlein,
W., and Fiders, G. (1981) Biochem. Biophys. Res. Commun. 101:599 606. The CTB protein is reacted with a 7-fold
molar excess of poly-L-lysine at pH 7.4 using 1-ethyl-3-(3-' Ihy' , .,,u~l) cali.-' ' present at 154 molar
excess over poly-L-lysine. After 24 hours, the ; ~ product is purified by chr~llldloy,_ h~, and titrated with
plasmid DNA using a gel retardation assay as described by Wu. Wu, G.Y., and Wu, C.H. (1987) J. Biol. Chem.
20 ~ 262:44294432.o The DNA:~otein co~plex is then used to fosm a CL~ C~ I; " app,."idl~ for the preferred mode
of delivery.
EXAMPLE 2. M ~ ~ l~
A DNA sequence cu,, O a strong promoter and a coding region encoding epitopes of the A8 and VDIV
proteins of Ch' , ' ll ,h li~ is c , ' ~ with CTB by the pGlyl~ method as ~1; - v~d above. A small
amount of Cholera toxin A ICTA) subunit is included in the cn m~û~ as an adjuvant. See Czt:H i~ky, C., and
Holmgren, J. (1995) The ~ r~ ~' 313:97-103; and Allen, J., Locksley, R.M., and Stephens, R.S. (1991) J.
Immunol. 147:674-679. The c~ is delivered once via vaginal ~ llGSPl\~y to ~' , ' free ~ . A
C~ ; ' free control group is given a placebo, ~npc~ n., y. At six one-week intervals, samples of serum and vaginal
mucus are taken from i ".;.h,a'5 of both groups. ELISA assays on serum samples show the presence of IgG specific
for A81VDIV in the t,edl"-~nl group and not in the control group. Similar assays on mucus sampies d~lllun:,lldl~ that
the ll~dl..._..l group has a high titer of slgA specific for A81VDIV while ". ' ' of the control group show no such
reaction. A s ' ~ ..l historical study of both groups shows a lower incidence of t-'/' ~/J;O i"ie~lions among
members of the ll~dllll~lll group compared with members of the control group.
EXAMPLE 3. ~--' ' ' ' ~l~n Reduced 1-" : y R~, e
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A DNA sequence cu"i a strong promoter and a chimeric coding region encoding a fusion of both CTB
and the heat shock protein ~HSP 57) of C~ /id lldC~ldilldt;.~ is Gom~ with CTB protein by the polylysine
method as d;~ ~cad above. The cU~ 1~~~ n is delivered once via vaginal s~rl~Qcitory to patients sufferiny from
Chl~, J/i i,,~.liuns and pelvic il,rla"""dldry disease (PID). A control group of similarly infected patients is given
5 a placebo ~u~,o~;~u.~. At six one-week intervals scratch tests and tine tests are pe~tu""ed on the forearms of
i"dN;duals of the two groups using whole ChlamJ~di~ Iysates. 'i-;d~.~ s of the two groups are also surYeyed for
d~ liun of the severity of PID a~ Jlulll~. The IlLallllLnl group displays a markedly ed 5~lla;l;ll;ly in
the scratch and tine tests, as well as ' d S~ lull~ of PID. The control group shows no such results.
10 EXAMPLE 4. r ~ t~,. S ~ and Reduced ~ y n ;e
A vaginal ~ ' y containing both the ONA -tlUL~ from Example 2 and the DNA r-a~ ~.u,.l from
Example 3 is ~ ~ ~~tPred to ~' : a infected with C~' ~Ji. A placebo ~ lu.y is . ' ~: ~d to an
GLIl~ d similar control group. The individuals of the ll~ai I group ~ H~nce both d~ as~d s~ .r of PID
and e "- ~e~ c..ll. "'~"tPd immunity to C!' 1dia. Mucus samples show high titers of slgA and IgG against
A8NDIV, but very little reaction to scratch and tine test using whole ChlamJ~di~ Iysates. The collateral DJ_: '
tissue" damage that is chdla~ lic of PID is therefore l~ There are no culll~làl ~ results among members
of the control group.
EXAMPLE 5. T~' of Or~an T. .'
A set of ~- : DNA plasmids is s .u~le;l each ~ ~ DNA encoding one of various MHC-1
proteins under control of a promoter. The resulting plasmid bank is chald~ ed and ' ~ d and
batches of each plasmid are :l 1 with CTB using the pol~ method as described above to create an organ
t H ~ i~ai~ ~ I,. I bank.
An organ donor is screened to d, l~, her MHC-1 gcaGIy~ The results of the screen are matched with
the d~upn, ;ale tH __ C , q from the bank d;~ s~d above.
A patient about to receive the donated organ is given doses of the '~ c~ ~o~:li-"~ as an atomized
di~ Ia;.~il via ' li., " as a rectal, ~l I o~ and orally as an enteric-coated tablet. The patient develops a rapid
oral; ' a,lce" to the foreign MHC-1 protein and displays a mllrh :" ' ~ ' e1 nonself ,, 1r. of the donated
organ. Because of the duration of e~nJI Lsa;ùl) and the abundant ~ ~ of the nonself MHC-1 gene in the mucosa-
ass~cialed Iymphoid tissues (MALT), the acquired tolerance is more long-lasting than would have been the case with
any more cu"~: ~ approach to oral t '~ i~aliuil using the MHC-1 protein itself, rather than the gene.
EXAMPLE 6. T-._ I of an A l Disease
Patients suffering from an autH ~ form of diabetes are orally ' : td a cnn-position C~Ua;alill!a
of a chimeric gene encoding both CTB and human insulin under control of a strong viral promoter wherein the DNA
of the chimeric gene was ' with CTB protein by polyl~s;lle as described above. The chimeric gene is
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delivered to the intestinal mucosa where it is hll~lllaii~d by cells of the MALT. These cells express a chimeric
fusion protein conc;~liu9 of CTB and insulin, which in combination effect a tolerization of the mucosal and system
immune system to the insulin epitopes. The patients' dulo:m.l~uiiy to insulin is " ' ~d and the progress of their
diabetes is slowed.
Other, non-lidbeli~., patients palli~,;,vdiillg in a long-term study are also ~ ' i~l~d the cGIll~.osi~ion. The
,e.~ " onset of diabetes among the voluul~ is lower than among individuals of an u~hdnl~ise similar control
group.
EXAMPLE 7. Gene Therapy
A cvl,~,o~il; n is prepared col~ !J of LTB and non mutant mRNA cGI~pon~ to the mutant gene that
is cauad~ of cystic fibrosis (CF). The cu~ o~ n is ~ ed twice daily to CF patients both orally and via
ildldldi The Co-~pG~ is localized to the mucosa of the lungs and the gdal~ ;llal tract where the enzyme
for which the CF patients are deficient is ~rliLi~ and lldll~i~nll~ produced. The ~lu~Jlu~ of CF in the patients
thus treated are much di
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