Note: Descriptions are shown in the official language in which they were submitted.
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1
TARGETED GENE TRANSFER USING G PROTEIN
COUPLED RECEPTORS
RELATED APPLICATION INFORMATION
This application claims the bene it of United States Provisional
Application No. 60/050,843 filed June 26, 1997.
STATEMENT OF FEDERAL SUPPORT
This invention was made with Government support under Grant No.
HL51818 from the National Institute of Health. The government has
certain rights in this invention.
~o FlELD OF THE INVENTION
This invention relates to methods and systems useful in the transfer
of nucleic acids into eukaryotic ceils.
BACKGROUND OF THE INVENTION
The capacities to introduce a particular foreign or native gene
sequence into a mammalian cell and to control the expression of that gene
are of substantial value in the fields of medical and biological research.
Such capacities provide a means for studying gene regulation, and for
designing a therapeutic basis for the treatment of disease.
2o The introduction of a particular foreign or native gene into a
mammalian host cells is facilitated first by introducing a gene sequence
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into a suitable nucleic acid vector. A variety of methods have.been
developed which are capable of permitting the introduction of such a
recombinant vector into a desired host cell. In contrast to methods which
involve DNA transformation or transfection, the use of viral vectors can
result in the rapid introduction of the recombinant molecule in a wide
variety of host cells. In particular, viral vectors have been employed in
order to increase the efficiency of introducing a recombinant nucleic acid
vector into host cells. Viruses that have been employed as vectors for the
transduction and expression of exogenous genes in mammalian cells
1o include SV40 virus (see, e.g., H. Okayama et al., Molec. Cell. Biol. 5,
1136-1142 (1985)); bovine papilloma virus (see, e.g., D. DiMaio et al.,
Proc. Natl. Acad. Sci. USA 79, 4030-4034 (1982)); adenovirus (see, e.g.,
J.E. Morin et al., Proc. Natl: Acad. Sci. USA 84, 4626 {9987)), adeno-
associated virus (AAV; see, e.g., N. Muzyczka et al., J. Clin. invest. 94,
1351 (1994)); herpes simplex virus (see, e.g., A.I. Geller, et al., Science
241, 1667 (1988)), and others.
Efforts to introduce recombinant molecules into mammalian cells
have been hampered by the inability of many cells to be infected by the
above-described viral or retroviral vectors. Limitations on retroviral
2o vectors, for example, include a relatively restricted host range, based in
part on the level of expression of the membrane protein that serves as the
viral receptor. M.P. Kavanaugh et al., Proc. Natl. Acad. Sci USA 91, 7071-
7075 (1994).
Accordingly, there exists a need in the art for improved methods of
introducing and expressing genes in target cells.
SUMMARY OF THE INVENTION
The shortcomings of current methods of receptor-mediated gene
transfer are overcome by the methods and complexes of the present
3o invention. In particular, the invention is based upon the unexpected
discovery by the present inventors that the rate limiting step of viral vector
uptake by cell surface receptors is not, as originally thought, the binding
event of the virus to the receptor, but rather the internalization of the
receptor itself. Accordingly, this invention relates to new complexes that
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facilitate the transfer of nucleic acids into eukaryotic cells. This invention
allows for targeting transfer vectors to specific cell types, attachment of
the
vectors to the cells and regulated cellular internalization of the vectors.
This invention comprises binding a transfer vector to a receptor that
is internalized by a cell. The receptor is one that is either internalized by
a
cell upon the cell's exposure to a specific ligand, or for which a receptor
may be induced to internalize by exposure to such a ligand.
BRIEF DESCRIPTION OF THE DRAWINGS
o Figure 1 is a schematic drawing illustrating a virus-receptor
complex of the present invention. This Figure illustrates an adenovirus
targeted to an internalizing seven transmembrane receptor.
Figure 2 is a schematic representation of particular embodiments of
~5 the present invention.
Figure 3 is a graphical representation of the Cf secretory
responses of human airway epithelia to lumenal NECA (A2b agonist),
isoproterenol, bradykinin, or ATP (all 10'~ M).
Figure 4A is a graphical representation of the dose-effect
relationship between bs-Ab concentration and gene transfer efficiency in
A9-null cells ( ~ ) compared with HA-P2Y2-A9 administered sequentially
(O) or as preformed conjugates (1).
Figure 4B is a graphical representation of a study to evaluate the
specificity of increased gene transfer with bs-Ab in A9-HA-P2Y2 and A9-
null cells pre-treated with specific or non-specific bs-Ab or after chronic
desensitization of HA-P2Y2 receptors by pretreatment with ATPyS.
Figure 5 is a graphical representation of gene transfer with bs-Ab in
null A9 and A9 cells expressing an HA-tagged BK" receptor.
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Figure 6 is a graphical representation of gene transfer in CHO cells
with bs-Ab to HA-tagged P2Y2 and ~i2 receptors and adenovirus fiber
protein.
Figure 7 is a graphical representation of biotin-UTP stimulation of
inositol phosphate formation in P2Y2 receptor expressing ( ~ ) but not wild-
type (~) astrocytoma cells.
Figure 8 is a graphical representation of the stimulation of gene
o transfer in A9 (wt) and HA-P2Y2-A9 cells in the presence of biotin-UTP
conjugated by streptavidin to biotin-Ad.
Figure 9A is a graphical representation of a comparison of agonist
potency of U2P4 and UTP in astrocytoma cells expressing P2Y2 receptors.
Figure 9B is a graphical representation of the metabolic stability of
U2P4 compared with UTP in cystic fibrosis sputum.
DETAILED DESCRIPTION OF THE INVENTION
2o The present invention now will be described more fully hereinafter
with reference to the accompanying drawings, in which preferred
embodiments of the invention are shown. This invention may, however,
be embodied in many different forms and should not be construed as
limited to the embodiments set forth herein. Rather, these embodiments
are provided so that this disclosure will fully convey the scope of the
invention to those skilled in the art. Like numbers refer to like elements
throughout.
A transfer vector-receptor complex of the present invention
comprises a transfer vector bound to a receptor that is capable of being
3o internalized into a cell. The transfer vector may contain an exogenous
nucleic acid sequence (e.g., a gene), and may express an exogenous
protein or peptide. In particular preferred embodiments, described in more
detail hereinbelow, the transfer vector is targeted to a seven
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transmembrane (7-TM) receptor by means of an antibody specific to the
receptor, by means of a peptide expressed by the transfer vector that
specifically binds said receptor, or by means of a natural or modified
ligand. The transfer vector may be any suitable vector, including a viral
s vector, a plasmid, an oligonucleotide, or RNAIDNA chimeric molecules, as
is described more fully hereinbelow. Interaction between the 7-TM
receptor and the targeted complex results in receptor complex
internalization, thereby introducing the heterologous nucleic acid carried
by the transfer vector into the cell where it is expressed.
Viral Transfer Vectors of the Present Invention
One embodiment of the invention is described with reference to
Figure 1. In this embodiment, a complex (i.e., a conjugate) of the present
invention comprises a viral vector 10 (which is illustrated in the figure as
1s an adenovirus) attached to a 7-TM receptor 20, which receptor 20 is
present on a cell surface 100. The viral vector 10 is attached to the 7-TM
receptor 20 by means of a bifunctional bridging antibody 30. The
bifunctional bridging antibody 30 is composed of one antibody 40 which
specifically binds the viral vector. The antibody 40 is chemically cross-
linked to antibody 50, which specifically binds the 7-TM receptor 20.
Although the viral vector 10 is illustrated as an adenovirus vector
(AdV), it will be understood that the present invention may also be
practiced with other viral vectors, including but not limited to human and
nonhuman retrovirus (i.e., Maloney virus such as Moloney Murine
2s Leukemia Virus and lentiviruses) vectors, adeno-associated virus (AAV)
vectors, and herpes virus vectors (Figure 2). The viral vectors of the
present invention may be attenuated viruses or may be rendered non-
replicative by any method known to one skilled in the art.
However, the use of adenoviruses as the vector is currently
so preferred.
The viral vectors of the present invention will have the capacity to
include exogenous nucleic acids. The delivery of the heterologous nucleic
acid facilitates the replication of the heterologous nucleic acid within the
target cell, and the subsequent production of a heterologous protein
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therein. A heterologous protein is herein defined as a protein or fragment
thereof wherein all or a portion of the protein is not expressed by the target
cell. A nucleic acid or gene sequence is said to be heterologous if it is not
naturally present in the wild type of the viral vector used to deliver the
gene into a cell (e.g., the wild-type adenovirus genome). The term
"nucleic acid sequence" or "gene sequence, " as used herein, is intended
to refer to a nucleic acid molecule (preferably DNA). Such gene
sequences may be derived from a variety of sources including DNA,
cDNA, synthetic DNA, RNA or combinations thereof. Such gene
o sequences may comprise genomic DNA which may or may not include
naturally occurring introns. Moreover, such genomic DNA may be
obtained in association with promoter sequences or poly-adenylation
sequences. The gene sequences of the present invention are preferably
cDNA. Genomic or cDNA may be obtained in any number of ways.
Genomic DNA can be extracted and purified from suitable cells by means
well-known in the art. Alternatively, mRNA can be isolated from a cell and
used to prepare cDNA by reverse transcription, or other means.
Standard techniques for the construction of the vectors of the
present invention are well-known to those of ordinary skill in the art and
2o can be found in such references as Sambrook et al., Molecular Cloning: A
Laboratory Manual 2nd Ed. (Cold Spring Harbor, NY, 1989). A variety of
strategies are available for ligating fragments of DNA, the choice of which
depends on the nature of the termini of the DNA fragments and which
choices can be readily made by the skilled artisan.
As will be appreciated by one skilled in the art, the nucleotide
sequence of the inserted heterologous gene sequence or sequences may
be of any nucleotide sequence. For example, the inserted heterologous
gene sequence may be a reporter gene sequence or a selectable marker
gene sequence. A reporter gene sequence, as used herein, is any gene
3o sequence which, when expressed, results in the production of a protein
whose presence or activity can be monitored. Examples of suitable
reporter genes include the gene for galactokinase, beta-galactosidase,
chloramphenicol acetyltransferase, beta-lactamase, etc. Alternatively, the
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7
reporter gene sequence may be any gene sequence whose expression
produces a gene product which affects cell physiology.
A selectable marker gene sequence is any gene sequence capable
of expressing a protein whose presence permits one to selectively
propagate a cell which contains it. Examples of selectable marker genes
include gene sequences capable of conferring host resistance to
antibiotics (e.g., puromycin, ampicillin, tetracycline, kanamycin, and the
like), or of conferring host resistance to amino acid analogues, or of
permitting the growth of bacteria on additional carbon sources or under
0 otherwise impermissible culture conditions. A gene sequence may be
both a reporter gene and a selectable marker gene sequence. The most
preferred reporter genes of the present invention are the IacZ gene which
encodes the beta-galactosidase activity of E. coli; and the gene encoding
puromycin resistance.
~ 5 Preferred reporter or selectable marker gene sequences are
sufficient to permit the recognition or selection of the vector in normal
cells.
In one embodiment of the invention, the reporter gene sequence will
encode an enzyme or other protein which is normally absent from
mammalian cells, and whose presence can, therefore, definitively
2o establish the presence of the vector in such a cell.
The heterologous gene sequence may also comprise the coding
sequence of a desired product such as a suitable biologically active
protein or polypeptide, immunogenic or antigenic protein or polypeptide, or
a therapeutically active protein or polypeptide. Preferably, the
25 heterologous gene sequence encodes a therapeutically active protein or
poiypeptide. In one particular preferred embodiment, the heterologous
gene sequence encodes the cystic fibrosis transmembrane conductance
regulator (CFTR) protein or biologically active analogs, fragments, or
derivatives thereof. Alternatively, the heterologous gene sequence may
3o comprise a sequence complementary to an RNA sequence, such as an
antisense RNA sequence, which antisense sequence can be administered
to an individual to inhibit expression of a complementary poiynucieotide in
the cells of the individual.
1-25-00 ~1~~~~PM ~ 5IM8A5-~ 819 953 9538~~ ~
9 ~ I 3~ ~
ENT BY:SIMBAS
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f.~cprenlon of the heteroloaous dens may prov~do Immunogenic dr
antlpANa protein or polypopptids to schleve an an#body rwponao, which
antlbodlee can be odiacted fioM an anirnsl In a body fluid such as blood,
serum or a1oltss.
It ts, of ovune, poe~Ibla to employ, se the Insw~sd heteroiopoua
gene sequenos, a glno sequence which already pos~rsses~a promoter,
Initiation sequence, or processing ssquenvo.
Non-YI~ Yeators at the Present enwhtlon
In olhsr prsi~eirsd embod~rrwnts, tho transfer vector Is nonwiral,
Other s~rtable vlcton InGude, but aro not limited to, ollponudeotidss
(Indudlng RNA. DNA, synthetic end mod~flad nuolela adds), plasmlds. Fund
RNAIDNA chlmvrin moiecuiee as de~tGrl~d by ~. Komotx,
oligonudv~de vdcbors Itlduds an4sensp oliponudoo#dvo and
otlponuoieot<dow that ~lrns~lon as nbotyrrats. The non-vlrill ttensEer wotors
of the proaent irNerdton are able to -induda ~snous r~ude~c actdv os
described herolnabvw with roepe4t bo virtl l~sorooro, Oilaonuoieotldes,
plaarnids, end RN~tDNA chimsdo moloa~ ott~ be synthesized or
produovd by any sulhbla m~had knawn In the art.
Hewn Trnnsm~rtblene R~loaptoea of !hs P~ss4n! imrenUon
Receptors wcoordinp tO the prlant tnven#on betanp bo the ferr~ily of
T-TM receptors. See psn~J~y, 8. Vltatson et al,, The Q-Pro~rlrr Linked
Receptor FsctaBook, Aaadvmlc Press, Now York (1894); U.B. Patertt No.
6,482,8# to Klng et at,
Those skHled in the srt vet a~pprrctats that T-TM reCSptotl ere O
protein c4upled reo~ptoro. Any mammailah G protafi onuplvd rooeptor,
and the nude'ro tad sequences encoding t>leae receptors, may bo
employed ~in prncdalng the present Inven~on. Examplso of each rserptars
inctude, but arcs not llrnitsd tp, dopamine receptors, musoarinia ahollne~lc
reaepoon3. Q-adrenerpfc rscopbate, opistl r~saep~or~t, t~nnablrrold
r~eosptora, ser~ooonin rooepbore, p.a~drener~gta reoeptcrs, dnd purindcepton.
'fhe term "reaepeo~' ss used haroirl,l~ fn~ended 1A enoompsss subtypes of
- w~
s ,~ ~ g x ~nroc ~ a ~r~. ~~, ...~,
~~t 'd a56r, '~~
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the named receptors, and mutants and homologs thereof, along with the
nucleic acid sequences encoding the same.
Preferably, the 7-TM receptor for use according to the present
invention is a purinoceptor (e.g., P2Y~, P2Y2, P2Y4, P2Ys and P2Y~~), an
adenosine receptor (i.e., A1, A2, and A3, and sub-types thereof), a
bradykinin receptor (e.g., BK, and BK"), or a ~i-adrenergic receptor (e.g.,
~3~, X32 and ~i3). Also preferred is the C5A complement receptor. More
preferred are the P2Y2, BK,i, A2B, X32, and C5A receptors, with the P2Y2
receptor being most preferred. Thus, ligands that may be used to carry
0 out the present invention include nucleotides, nucleosides, catecholamines
(e.g., dopamine, 5-hydroxytryptophan), CSA, and bradykinin(s).
The P2Y2 (also known as the P2~)- purinoceptor undergoes
internalization upon activation with ATP, UTP and analogs thereof. These
receptor-types are abundant in number on the lumenal surface of the
human respiratory epithelium. Mason, S. J., et al. 1991. Br. J. PharmacoG
103, 1649-1656. Molecular conjugation of AdV to P2Y2-receptors, followed
by activation of these receptors by ATP/UTP, leads to internalization of the
vector-ligand-receptor complex into endosomes and thus provide an
alternative entry pathway for AdV into the WD epithelium, and thereafter to
2o gene expression.
Antibodies of the Present Invention
As shown in Figure 1 and Figure 2, one strategy for targeting
transfer vectors carrying heterologous nucleic acids to 7-TM receptors for
internalization into the cell is with a bispecific bridging antibody. In
general, the bispecific antibody is directed against epitopes on both the
transfer vector and the 7-TM receptor of interest (i.e., has a combining
region that specifically recognizes the transfer vector and a combining
region that specifically recognizes the 7-TM receptor), thereby forming a
"bridge" between the transfer vector and the receptor. Binding of the
bispecific bridging antibody to the 7-TM receptor induces internalization of
the receptor. The bound antibody-transfer vector complex is internalized
along with the 7-TM receptor, thereby introducing the transfer vector
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carrying the heterologous nucleic acid into the cell. According to this
embodiment of the invention, the transfer vector is preferably a viral
vector, more preferably, AdV, and the bispecific antibody comprises a
monoclonal antibody directed against the fiber (knob) protein of the
s adenovirus.
The term "antibodies" as used herein refers to all types of
immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. Of these, IgM and
IgG are particularly preferred. The antibodies may be monoclonal or
polyclonal and may be of any species of origin, including (for example)
~ o mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. See,
e.g., M. Walker et al., Molec. Immunol. 26, 403-11 (1989). The antibodies
may be recombinant monoclonal antibodies produced according to the
methods disclosed in Reading U.S. Patent No. 4,474,893, or Cabilly et al.,
U.S. Patent No. 4,816,567. The antibodies may also be chemically
constructed by specific antibodies made according to the method
disclosed in Segel et al., U.S. Patent No. 4,676,980.
Antibodies may be polyclonal or monoclonal, with monoclonal being
preferred. In particular embodiments, the antibodies are bridging
antibodies that are specific to both the target receptor and the transfer
2o vector. According to this embodiment, the bridging antibody is preferably
a monoclonal antibody directed to the adenovirus fiber (knob) protein.
Also preferred are monoclonal antibodies and bridging antibodies
comprising monoclonal antibodies that are directed to specific epitopes of
the 7-TM receptor of interest.
2s Antibodies that bind to the epitope (i.e., the specific binding site)
that is bound by the antibody to the 7-TM receptor can be identified in
accordance with known techniques, such as their ability to compete with
labeled antibody to the 7-TM receptor in a competitive binding assay.
Antibody fragments included within the scope of the present
3o invention include, for example, Fab, F(ab')2, and Fc fragments, and the
corresponding fragments obtained from antibodies other than IgG. Such
fragments can be produced by known techniques.
Polyclonal antibodies used to carry out the present invention may
be produced by immunizing a suitable animal (e.g., rabbit, goat, etc.) with
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an antigen to which a monoclonal antibody to the 7-TM receptor binds,
collecting immune serum from the animal, and separating the polyclonal
antibodies from the immune serum, in accordance with known procedures.
Monoclonal antibodies used to carry out the present invention may
s be produced in a hybridoma cell line according to the technique of Kohler
and Milstein, Nature 265, 495-97 (1975). For example, a solution
containing the appropriate antigen may be injected into a mouse and, after
a sufficient time, the mouse sacrificed and spleen cells obtained. The
spleen cells are then immortalized by fusing them with myeloma cells or
o with lymphoma cells, typically in the presence of polyethylene glycol, to
produce hybridoma cells. The hybridoma cells are then grown in a
suitable media and the supernatant screened for monoclonal. antibodies
having the desired specificity. Monoclonal Fab fragments may be
produced in Escherichia coli by recombinant techniques known to those
s skilled in the art. See, e.g., W. Huse, Science 246, 1275-81 (1989).
Antibodies specific to the 7-TM (e.g., P2Y2 ) receptor: may also be
obtained by phage display techniques known in the art.
Those skilled in the art will be familiar with numerous specific
immunoassay formats and variations thereof which may be useful for
2o carrying out the method disclosed herein. See generally E. Maggio,
Enzyme-Immunoassay, (1980)(CRC Press, Inc., Boca Raton, FL); see
also U.S. Patent No. 4,727,022 to Skold et al. titled "Methods for
Modulating Ligand-Receptor Interactions and their Application," U.S.
Patent No. 4,659,678 to Forrest et al. titled "Immunoassay of Antigens,"
2s U.S. Patent No. 4,376,110 to David et al., titled "Immunometric Assays
Using Monoclonal Antibodies," U.S. Patent No. 4,275,149 to Litman et al.,
titled "Macromolecular Environment Control in Specific Receptor Assays,"
U.S. Patent No. 4,233,402 to Maggio et al., titled "Reagents and Method
Employing Channeling," and U.S. Patent No. 4,230,767 to Boguslaski et
3o al., titled "Heterogenous Specific Binding Assay Employing a Coenzyme
as Label." Applicants specifically intend that the disclosures of all U.S.
Patent references cited herein be incorporated herein by reference.
Antibodies as described herein may be conjugated to a solid
support suitable for a diagnostic assay (e.g., beads, plates, slides or wells
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formed from materials such as latex or polystyrene) in accordance with
known techniques, such as precipitation. Antibodies as described herein
may likewise be conjugated to detectable groups such as radiolabels (e.g.,
355 ~2s1, ~3~1), enzyme labels (e.g., horseradish peroxidase, alkaline
s phosphatase), and fluorescent labels (e.g., fluorescein) in accordance with
known techniques. The term "antigenic equivalents" as used herein, refers
to proteins or peptides which bind to an antibody which binds to the protein
or peptide with which equivalency is sought to be established. Antibodies
which are used to select such antigenic equivalents are referred to as
"selection antibodies" herein.
Non-Antibody Based Targeting Strategies
The invention has been described above with respect to bispecific
bridging antibodies as means of targeting the transfer vector to the 7-TM
~ s receptor for internalization. As shown in Figure 2, alternative targeting
strategies include those utilizing peptides and 7-TM receptor
agonistlantagonists.
With respect to peptides, the peptide can be a natural ligand that
binds to the 7-TM receptor. Peptide agonists and antagonists of 7-TM
2o receptors are known in the art. Additionally, novel 7-TM receptor
agonists/antagonists can be identified as described by U.S. Patent No.
5,482,835 to King et al. Alternatively, the peptide can be identified by
phage display techniques, or any other method in the art, as binding to the
7-TM receptor.
2s Methods of synthesizing or producing peptides are welt-known in
the art. In one particular embodiment, nucleic acids encoding the peptide
are fused to or inserted into the gene encoding the AdV knob protein, such
that a knob-peptide chimeric protein is expressed. It is known, for
example, that exogenous nucleic acid can be expressed in the C-terminus
30 or the H1 loop region of the knob protein. In alternate embodiments,
concatomers of the peptide are expressed in the knob protein.
As a further alternative, targeting can be achieved with peptides
incorporated into "receptorbodies". In general, a receptorbody is a
truncated receptor in which a peptide that binds to a 7-TM receptor is
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substituted for the intracellular region of the receptor. For example, a
truncated BK" receptor can be fused to a peptide ligand for a 7-TM
receptor. This complex will bind to a recombinant AdV expressing the
bradykinin peptide in the knob protein region by way of the truncated
s receptor. The peptide ligand fused to the truncated BK" receptor will then
target this complex to its cognate receptor on the cell surface.
As yet a further alternative, the transfer vector can be targeted to
the 7-TM receptor by a chemically-linked high affinity agonistlantagonist of
the 7-TM receptor. High-affinity agonists/antagonists may be peptide
o ligands, as described above. Alternatively, they are other molecules, such
as ATP, UTP, dinucleotides (described in more detail hereinbelow), and
derivatives thereof.
In one particular embodiment, a biotin (B) -UTP is used as a
targeting agonist for the P2Y2 receptor. The B-UTP can interact with a
s biotinylated viral transfer vector in the presence of streptavidin {SA) to
give
a virus-biotin-SA-biotin-UTP complex that will be targeted to the P2Y2
receptor. Alternatively, oligonucleotides, plasmids, and RNA/DNA
chimeric molecules can be synthesized or produced to incorporate B-UTP
or any other suitable labeled nucleotide.
2o In a further alternative embodiment, high affinity agonists or
antagonists can be directly linked to the transfer vector using sulfo-N-
hydroxysuccinimide (NHS) as described in more detail below.
:5IM8A5 ; CA 0229s31s 1999-i2-is gIMBAS-~
1-25-00 :12~28PM ; 819 953 9538~~ 8
SENT BY
' ,.. --, ~! ~ ~ ,~ a N X999
divtln and Covehnt Cor~u~sbr
Fxomptl~ of compotJnda that can bs used to carry out the pnaent
invention include thosa havlnd the qenent tonnuls; ,.
. .
A -O H
n
wheroin:
A is a pu~ine or pyrlmidlne boas te.a., adenine, guanine, thymine,
cyDoslns, undt)~sach patina or pytimldlna bass is proferabiy ~otr~ed to the
ribose or deoxyriboaa tins by cavalant bend bo the 6 nftrogetr in the oase of
purinle, of by oovsietlt bond to the 1 nltcogen fn the ce~e of pyrtrrddlnea):
R, le H or OH; ~rtsct n la from t to 8, pnrferebly 9 or 4. The trsna~r veceor
to oovalently or nonoovalandy )olned or cor~upAted bo the patina or
pyrtmidlne base, or the corroapondlng ribdre or deoxyriboea rir~~, by any
suitable means, ouch at by aov~tienHy jolr~lng a llnldnp potynror chain
therrbo in dny sultobio position (e,p.. a rlr>g c11'bon ouch as the 5 carbon
(n
a pyrlmidtne, or the ~ or 8 oacbott in r purlne~, bo whloh ilnldnd group the
llgend mery be oovtter~t~r allac~ed, ortD which tinidr~ group w blaNn prplfp
may be athched, with a bbtin group oo~tarrby ~otr»d eo iha lig$nd (aes
below) and tha twp blotM groups jobled to ona another by means of an
avidtn proWp to whtct~ both biotin groups ana joined or conjugated.
9ped~c examples Of lipsnds that ~n bo ufed to carry out tha
prr4int lrwer~tion lnotude thoaa having the general formula;
whsretn~.
14
~. ,..-...-.i,v ~a..,~99st ~a~L... . .. s ~ s s R >iareE~s 66st ~t:a ~N~r
CA 02295315 1999-12-15
WO 99/00511 PCT/US98/13336
A and B are each independently a purine or pyrimidine base (e.g.,
adenine, guanine, thymine, cytosine, uracil); preferably, A is uracil and B is
cytosine;
R~ and R2 are each independently selected from the group
5 consisting of H or OH;
n is from 1 to 6, preferably 3 or 4; and
said transfer vector is covalently or noncovalently conjugated or
joined to A or B or the ribose or deoxyribose ring to which A or B is joined,
either directly or indirectly by means of a linking group, in the same
o manner as described above.
Linking groups used to cant' out the present invention are, in
general, polymers, including both water soluble polymers and water
insoluble. Water soluble, or hydrophilic, linking groups are preferred. The
polymers are elongate flexible chains of repeating monomeric units, and
s may carry or contain functional groups along the chain length thereof.
Numerous polymers that can be functionalized to function as. linking
groups for the ligand and the vector, typically by a covalent bond, are
known, and will be readily apparent to those skilled in the art. Examples
include, but are not limited to, polysaccharides such as dextran, polyvinyl
2o alcohol, polypeptides such as polylysine, and polyacrylic acid. The ligand
and the vector may be bound to the linking group in any conformation or
position, including to the free chain end thereof. In general, the linking
group wilt comprise a chain of from 2 to 24 carbons, optionally substituted
as described above.
2s Biotin can be covalently joined to the ligand by conventional
techniques and both biotin groups joined to an avidin or strep-avidin group
in accordance with known techniques to form a conjugate of the vector
and the ligand.
CA 02295315 1999-12-15
WO 99/00511 PCT/US98/13336
16
Examples of ligands to which biotin is covalently joined include:
0
HN~NH 0
H H
NH
SI O
wherein R2 is H or OH, preferably OH; and n is equal to 1 to 4, preferably
3. Such compounds are known and commercially available. The uridine
group shown can be replaced with another purine or pyrimidine base as
described above, with the biotin and linking polymer chain shown between
the biotin group and the uridine group above covalently joined to the
~ 5 purine or pyrimidine base in any suitable position (e.g., a ring carbon
such
as the 5 carbon in a pyrimidine, or the 2 or 8 carbon in a purine).
Significantly, an oligonucleotide (e.g., a DNA, RNA, or chimera of 5 or 10
to 30 or 50 bases) can be synthesized with one or more bases conjugated
to a biotin in this manner, and the thus biotinylated oligonucleotide
2o conjugated to a biotinylated ligand as described herein by means of an
avidin.
A biotin group can be covalently joined to the vector (particularly
vectors having free amine groups such as viral vectors) by means of the
EZ-LINKT"" Sulfo-NHS-LC-Biotinylation Kit, available from Pierce (3747 N.
25 Meridian Road, P.O. Box. 117, Rockford, IL fi1105). An example of a
compound that can be used to biotinylate a primary amine on the vector is
Sulfo-NHS-LC-Biotin, available from Pierce, and having the structure:
0
O ~' _NH
30 Na03S
O O H H
II II
O-C-(CHz)5-NH-C-(CHZ)4 S
O
CA 02295315 1999-12-15
WO 99/00511 PCT/US98/13336
17
In an alternate embodiment, the biotin group shown in the sulfo compound
described above can be removed and replaced with a covalent linkage to
a ligand, as described above, to provide a direct covalent linkage from the
vector to the ligand.
Target Cells of the Present Invention
Hematopoietic stem cells, lymphocytes, vascular endothelial cells,
respiratory epithelial cells, keratinocytes, skeletal and muscle cardiac
cells,
neurons and cancer cells are among proposed targets for therapeutic
o gene transfer, either ex vivo or in vivo. See, e.g., A.D. Miller, Nature
357,
455-460 (1992); R.C. Mulligan, Science 260, 926-932 (1993). These cells
and other eukaryotic cells are suitable target cells for the vectors and
methods of the present invention. One advantage of the present invention
is that it can be used to target heterologous nucleic acids to cells that do
s not usually bind the transfer vector, i.e, a virus vector.
In particular, any cell that expresses a receptor from the 7-TM
receptor family is a suitable target for use according to the present
invention. Preferred are cells that express purinoceptors (e.g., P2Y~,
P2Y2, P2Y4, P2Ys, P2Y»), adenosine receptors (i.e., A1, A2, A3),
2o bradykinin receptors (e.g., BK, , BK"), or (i-adrenergic receptors (e.g.,
(i~,
~2, ~3). More preferred are cells that express P2Y2, BK", A2B, ~Z, Csa,
receptors, with cells that express the P2Y2 receptor being most preferred.
Also preferred as targets are respiratory epithelial cells, particularly
differentiated columnar airway epithelial cells. The cells may be
25 administered the conjugate in vitro or in vivo, such as by administration
of
an aerosol containing the conjugate to the luminal surface of airway
epithelial cells.
Gene Transfer Technology
3o The methods of the present invention provide a means for
delivering heterologous nucleic acid independent of the target cell nucleus
into a broad phylogenetic range of host cells. The vectors, methods and
pharmaceutical formulations of the present invention are additionally
CA 02295315 1999-12-15
WO 99/00511 PCT/US98/13336
18
useful in a method of administering a protein or peptide to a subject in
need of the desired protein or peptide, as a method of treatment or
otherwise. in this manner, the protein or peptide may thus be produced in
vivo in the subject. The subject may be in need of the protein or peptide
s because the subject has a deficiency of the protein or peptide, or because
the production of the protein or peptide in the subject may impart some
therapeutic effect, as a method of treatment or otherwise, and as
explained further below.
The gene transfer technology of the present invention has several
applications. The most immediate applications are perhaps in elucidating
the processing of peptides and functional domains of proteins. Cloned
cDNA or genomic sequences for proteins can be introduced into different
cell types in culture, or in vivo, in order to study cell-specific differences
in
processing and cellular fate. By placing the coding sequences under the
s control of a strong promoter, a substantial amount of the desired protein
can be made. Furthermore, the specific residues involved in protein
processing, intracellular sorting, or biological activity can be determined by
mutational change in discrete residues of the coding sequences.
Gene transfer technology of the present invention can also be
2o applied to provide a means to control expression of a protein and to
assess its capacity to modulate cellular events. Some functions of
proteins, such as their role in differentiation, may be studied in tissue
culture, whereas others will require reintroduction into in vivo systems at
different times in development in order to monitor changes in relevant
2s properties.
Gene transfer provides a means to study the nucleic acid
sequences and cellular factors which regulate expression of specific
genes. One approach to such a study would be to fuse the regulatory
elements to be studied to reported genes and subsequently assaying the
3o expression of the reporter gene.
Gene transfer also possesses substantial potential use in
understanding and providing therapy for disease states. There are a
number of inherited diseases in which defective genes are known and
have been cloned. In some cases, the function of these cloned genes is
CA 02295315 1999-12-15
WO 99/00511 PCT/US98I13336
19
known. In general, the above disease states fall into two classes:
deficiency states, usually of enzymes, which are generally inherited in a
recessive manner, and unbalanced states, at least sometimes involving
regulatory or structural proteins, which are inherited in a dominant manner.
s For deficiency state diseases, gene transfer could be used to bring a
normal gene into affected tissues for replacement therapy, as well as to
create animal models for the disease using antisense mutations. For
unbalanced disease states, gene transfer could be used to create a
disease state in a model system, which could then be used in efforts to
o counteract the disease state. Thus the methods of the present invention
permit the treatment of genetic diseases. As used herein, a disease state
is treated by partially or wholly remedying the deficiency or imbalance
which causes the disease or makes it more severe. The use of site-
specific integration of nucleic sequences to cause mutations or to correct
~ 5 defects is also possible.
In one particularly preferred embodiment, the present invention is
employed to express an exogenous CFTR protein in respiratory
epithelium. According to this embodiment, it is preferred to use an AdV
transfer vector canying the CFTR gene. The AdV-CFTR is directly linked
2o to the dinucleotide UP4C by a sulfo-NHS linker, as described above.
Binding of UP4C to the P2Y2 receptor on the apical surface of the
respiratory epithelium will induce internalization of the entire UP4C-sulfo-
NHS-AdV-CFTR complex into epithelial cells.
2s Pharmaceutical Formulations, Subjects, and Methods of
Administration
Suitable subjects to treated according to the present invention
include both avian and mammalian subjects, preferably mammalian. Any
mammalian subject in need of being treated according to the present
3o invention is suitable. Human subjects are preferred. Human subjects of
both genders and at any stage of development (i.e., neonate, infant,
juvenile, adolescent, adult) can be treated according to the present
invention. Human subjects afflicted with cystic fibrosis are preferred.
CA 02295315 1999-12-15
WO 99/00511 PCT/ITS98/13336
Active compounds of the present invention may be prepared as
pharmaceutically acceptable salts thereof. Pharmaceutically acceptable
salts are salts that retain the desired biological activity of the parent
compound and do not impart undesired toxicological effects. Examples of
s such salts are (a) acid addition salts formed with inorganic acids, for
example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric
acid, nitric acid and the like; and salts formed with organic acids such as,
for example, acetic acid, oxalic acid, tartaric acid, succinic acid, malefic
acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid,
~o benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,
naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,
naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (b)
salts formed from elemental anions such as chlorine, bromine, and iodine.
Active compounds of the present invention can be administered to
~5 a subject in need thereof by any suitable means including oral, rectal,
transmucosal, topical or intestinal administration; parenteral delivery,
including intramuscular, subcutaneous, intramedullary injections, as well
as intrathecal, direct intraventricular, intravenous, intraperitoneal,
intranasal, or intraocular injections. Alternately, one may administer the
2o compound in a local rather than systemic manner, for example, in a depot
or sustained release formulation. Administration to the lungs is preferred.
Active compounds disclosed herein may be administered to the
lungs of a subject by any suitable means, but are preferably administered
by administering an aerosol suspension of respirable particles comprised
of the active compound, which the subject inhales. The respirable particles
may be liquid or solid.
Aerosols of liquid particles comprising the active compound may be
produced by any suitable means, such as with a pressure-driven aerosol
nebulizer or an ultrasonic nebulizer. See, e.g., U.S. Pat. No. 4,501,729.
so Nebulizers are commercially available devices which transform solutions
or suspensions of the active ingredient into a therapeutic aerosol mist
either by means of acceleration of compressed gas, typically air or oxygen,
through a narrow venturi orifice or by means of ultrasonic agitation.
Suitable formulations for use in nebulizers consist of the active ingredient
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M a flqu(d canter, the rdhrs InQnedient oomprtslng up to d0~ wlw of
the tontntadon, but pndvrsbty leas than 20% wlw. 'frie carrlor to typlcaily
water (and moat prefrnbly :~rtle, pyropenfrgs avatar) ar a dil~rte e~qwous
a~cahoiic ~otutlon, pnierebly mivda (eatontc with body ~ulds by the e;ddftfon
oi, ibr example, wdlum chloride,
Optiorwl addl'ttvvss Include prrservatlves if the i~ormuiabon Is not
rnwda a~tarlie, for example, mettrr! hydroxybstuo~, arrtlaxldants, flavoring
agenb, volat0e oils, 6utferlng agents and surfe~ctants.
Aen~ots of solid psrbdee oomprlsihQ tha active compound may
likewise be produced w~h any solid particulate medicament aerosol
penenbor. Aerosol peneratoro fur rdmlnlatsrlng solkl pertloulate
rnedioamsnb to a eubjeot produce pat~des nrhtoh are n~pirabN, ae
explained above, and 9eneraEs a v4lums of aerosol oonteintnp a
prodetirMined metered doae of a ttbdbprnon! st s raita sultsble for human
e~dminislntion. pre IUusttatlva type of eoikf paWculabr aerosol penorator is
an lnsulAshor. 8ultsble lbrtnuiwtione for admlnfstratfon by lneufrtatlon
indude ilnsly commlnuhd powder8 which fist' be delivered by msarn
o~ an lnsufrie~r or taksa Into the nasal aavlty in the manner of a Bnuif, !n
the Irreuftlebr, the powder (a.g" a metered down thereof ethcttvs to carry
out the treatments described her~)'is cvrtatnsd In ~poulea or cerMdpos,
typiaatly mode of pslaln or plse~c, which sfie eftllar plarasd or opendd In
Htu and the powdeN 4eNwrod by air drawn thro~t~h the device upon
ihhalatbn or by moans of t manuatly.oper~ated pump, 'rha powder
err~pioysd tn the ineufiatipr consists either svlvly df the actiVS Ingrvdfsnt
or
of a powder blend cornpttslr~ the ectlvl ir~rsdteht, a suitable powder
dlluer~t, suoh as lactose, and an optional surlbdartt, The ectfvs lngrodlent
typioalty comprises from 0.~ to X40% wlw of the iotmufation. A second
type of lltuitnwthro eet~otd generator vomprtsec a rhstArsd dose inhslsr.
Mot~rod dose inhalant an pr~au~'I~ed aet4sol dhpenssro, typioolly
containing s vuspemion or aolutlan ib~rnulstlon of the active In~radienf an a
Aqulfled propelisM. During use thtse devices dlscharpe the ibmuiletion
throtrQh a valve adapts ~ air s metered volumr, typically
from 'i 0 to 150 pl, to p~oduoe a Tlnepsrfk~ spray ovntalntnQ the active
Z1 - ,...
.,~.-.._..gt 'd.~9~BL 'OlI S ; S a lI AY9~ ~8 BRRI 'f7 'Writ
CA 02295315 1999-12-15
WO 99/00511 PCT/US98/13336
22
ingredient. Suitable propellants include certain chlorofluorocarbon
compounds, for example, dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane and mixtures thereof. The formulation may
additionally contain one or more co-solvents, for example, ethanol,
s surfactants, such as oleic acid or sorbitan trioleate, antioxidants and
suitable flavoring agents.
The aerosol, whether formed from solid or liquid particles, may be
produced by the aerosol generator at a rate of from about 10 to 150 liters
per minute, more preferably from about 30 to 150 liters per minute, and
most preferably about fi0 liters per minute. Aerosols containing greater
amounts of medicament may be administered more rapidly.
The dosage of the active compounds disclosed herein or
pharmaceutically acceptable salt thereof, will vary depending on the
condition being treated and the state of the subject, but generally may be
an amount sufficient to achieve dissolved concentrations of active
compound on the airway surfaces of the subject of from about 10'' to
about 10 '3 Moles/liter, and more preferably from about 10'6 to about 3 x
10'~ Moles/liter. Depending upon the solubility of the particular fomlulation
of active compound administered, the daily dose may be divided among
20 one or several unit dose administrations. Other compounds may be
administered concurrently with the active compounds, or salts thereof, of
the present invention.
Solid or liquid particulate pharmaceutical formulations containing
active agents of the present invention should include particles of respirable
2s size: that is, particles of a size sufficiently small to pass through the
mouth
and larynx upon inhalation and into the bronchi and alveoli of the lungs. In
general, particles ranging from about 1 to 5 microns in size (more
particularly, less than about 4.7 microns in size) are respirable. Particles
of
non-respirable size which are included in the aerosol tend to be deposited
3o in the throat and swallowed, and the quantity of non-respirable particles
in
the aerosol is preferably minimized. For nasal administration, a particle
size in the range of 10-500 ~,m is preferred to ensure retention in the nasal
cavity.
CA 02295315 1999-12-15
WO 99/00511 PCT/US98/13336
23
In administering the active compounds of the present invention,
they may be administered separately (either concurrently or sequentially)
or, alternatively and preferably, they may be pre-mixed and administered
as preformed conjugates. As an illustrative example, as suitable dose of a
s transfer vector carrying a heterologous nucleic acid of interest, can be pre-
mixed with a targeting molecule (i.e., a bispecific bridging antibody, a
peptide, biotin-UTP, etc.) and the complex administered to the subject.
In the manufacture of a formulation according to the invention,
active agents or the physiologically acceptable salts or free bases thereof
o are typically admixed with, inter alia, an acceptable carrier. The carrier
must, of course, be acceptable in the sense of being compatible with any
other ingredients in the formulation and must not be deleterious to the
patient. The carrier may be a solid or a liquid, or both, and is preferably
formulated with the compound as a unit-dose formulation, for. example, a
~s capsule, which may contain from 0.5% to 99% by weight of the active
compound. One or more active compounds may be incorporated in the
formulations of the invention, which formulations may be prepared by any
of the well-known techniques of pharmacy consisting essentially of
admixing the components.
2o Compositions containing respirable dry particles of active
compound may be prepared by grinding the active compound with a
mortar and pestle, and then passing the micronized composition through a
400 mesh screen to break up or separate out large agglomerates.
The pharmaceutical composition may optionally contain a
25 dispersant which serves to facilitate the formation of an aerosol. A
suitable
dispersant is lactose, which may be blended with the benzamil or phenamil
in any suitable ratio (e.g., a 1 to 1 ratio by weight).
In summary, the transfer vectors of the present invention can be
used to stably transfect either dividing or non-dividing cells, and stably
so express a heterologous gene. Using this vector system, it is now possible
to introduce into dividing or non-dividing cells, genes which encode
proteins that can affect the physiology of the cells. The vectors of the
present invention can thus be useful in gene therapy for disease states, or
for experimental modification of cell physiology.
CA 02295315 1999-12-15
WO 99/00511 PCT/US98/13336
24
Having now described the invention, the same will be illustrated
with reference to certain examples which are included herein for illustration
purposes only, and which are not intended to be limiting of the invention.
EXAMPLE 1
Models of Human Airway Epithelium
Epithelial cells are derived from CF and non-CF nasal and
bronchial airway epithelia using procedures similar to those described by
Gray et al . 1996. Am. J. Respir. Cell Mol. Biol. 14, 104-112. Resected
to nasal turbinates or portions of mainstem/lobar bronchi representing excess
donor tissue are obtained at the time of lung transplantation under the
auspices of the University of North Carolina at Chapel Hill Institutional
Committee on the Protection of the Rights of Human Subjects. Epithelial
cells are removed from the specimens by protease XIV digestion as
15 described (Wu, R., et al., 1985. Am. Rev. Respir. Dis. 132, 311-320), but
omitting the filtration step. 1-2 x 106 cells are plated per 100mm tissue
culture dish in modified LHC9 medium. Lechner, J. F. and Laveck, M. A.
1985. J. Tiss. Cull. Meth. 9, 43-48. The modifications include increasing
the EGF concentration to 25nglml, adjusting the retinoic acid concentration
2o to 5x 10'6M, and supplementation with 0.5 mg/ml bovine serum albumin
and 0.8% bovine pituitary extract. At approximately 75% confluence, the
cells are harvested by trypsinization and passage 1 cells are plated at a
density of 2.5x105 cells on Transwell-Col inserts (Corning-Costar, 24mm
Qs, 0.4Nm pore size), in modified medium. The medium is similar to the
25 supplemented LHC9 except that a 50:50 mixture of LHC Basal (Biofluids)
and DMEM-H is used as the base, amphotericin and gentamycin are
omitted and the EGF concentration is reduced to 0.5 ng/ml. After the cells
grow to confluence (4-6 days) the apical surface of the cultures are given
an air-liquid interface for another 25-30 days until use.
3o Initial histological analyses of human WD cultures derived from non
CF nasal airways after 34 days of culture indicate that the epithelium is
pseudostratified mucociiiary, with abundant cilia and cell-types
representative of those present in human nasal airways in vivo.
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EXAMPLE 2
Esven Trsntrriernbratto ReQeptor
Expntston on tho Api~l Merrrtrrane of WD Galls .
InveetiQaNo~ were eanied outto Idetd9f~ whloh 7-TM rocaptpra, if
any, are fooal~ed fn the epleel membrane of WD human ainwy aplthafGal
cells. Culturoa of WD calls wen axposrd to NECA (an Aa, receptor
agvntat), Isaproteronol, brsuiyklnln, yr ATP (sill at l O~My, and Cl. seastory
reeponelvenesa was determined. A: shown in l~tEuro S, a Ci- tecretory
reepvnse was detested In thv presence o~ all of the egonisns, howswr, the
qroataet response was observed In the presonoe of ATP. Thees results
strongly eugpast that tut~ottona! adenoelns, ~-adron~gtc, btadyldnin and
purino rsceptots sro proeont on the pptdsl surfeos et airway epithelia.
EXAMP~ 3
Bindbr~ and M~rt~allsation of AdV
in Hu~tt~n PD and WD ettwsy optthilial sells
!=xporimsnts ehowlnn that e,denavtrue wcbor (/~dV~lnternadi~atbn.
not Adll-binding, !s the ribe~tlmltlr~ stop rpauiHnp In low ailldency ~sne
transfior to RTE 1Np cutturos oro nspestod~~ with human arkuras to
datermlne !t the same nste~.Ilmltlnp siJep Is prnant, if thls !s hideed ihs
cast, then sltflough the oohs heave ~ raduc~ed rubs of Ins~ltranon, it rn.y
be possible to Incroaee pane ~rsnil~sr oficlency to WG cuiiures by
enhartr~np the amount of AdV that binds tO see cuidare~rpee, For a
given concentnatlon of AdV, sxnoead tv ~dher PD or WD cuftunea, only
approxirnstly 0,!-t 94 of the fate! AdV eaeposed to cavils remains attsohad
slaver waehlne. Enhanaemant of AdV-blndinp stow that schivwed with s
single aaposura taa~l8 tp rn incxuase In pohe transfer, since Increasing the
blndlnd of AdV to veils will increase the pl~babtl'~ty thAt 4n Mtert~N~bon
event Iw~da to AdV entry.
To determine the nltealmitfng slap fbr inetfiolent gins kanffer In
human vultures, PD and YYD vunurrs era wcpovad by s°8.-I1d51lLacZ (1.2
x
101° p) for analyses of AdV.blndipp, ltredort and tranagane
~Ri '~ ~aRl, vu t ~ s a a rwAf:a ~aRr ~r~ v~~
CA 02295315 1999-12-15
WO 99/00511 PCT/US98/13336
26
expression in the human cultures. To investigate the effect of increasing
the concentration of AdV and/or the duration of exposure to AdV, PD and
WD cultures are exposed to 35S-AdSVLacZ {Pickles, R. J., et al., 1996.
Human Gene Therapy 7, 921-931 ) at a range of concentrations (10'-10'2
plml) for a number of time points (1-24hrs) at 4°C, after which
cultures are
washed in medium and then divided into three groups for analyses.
Binding is measured as cell-associated radioactivity. internalization of
bound AdV is measured by transferring the cultures to 37°C for 6 hrs
followed by measurement of cell-associated radioactivity after removal of
non-internalized radioactivity. Expression is measured by transferring the
cultures to 37°C for 48 hrs before measuring a-gal activity.
Radioactive
counts per minute (CPM) and ~3-gal activity are standardized with respect
to the nominal surface area of the culture surface because the apical
surface area of cells exposed to vector is the most appropriate
denominator, as it allows direct comparison to the epithelium in vivo.
It is likely that with PD cells, for a specific incubation time, a 10-fold
increase in concentration will result in a 10-fold increase in AdV
attachment, internalization and gene expression as tong as saturation of
receptor uptake and expression systems does not occur. With WD
2o cultures, although a 10-fold increase in AdV attachment is expected, the
corresponding 10-fold increases in internalization and expression are not.
These data indicate that increased binding alone does not overcome the
rate-limiting step (internalization) into WD cultures.
EXAMPLE 4
Targeting of AdV Vectors to the P2Y2 Receptor
To test the concept that the P2Y2 receptor is a candidate receptor
for targeting based on the ability to bind and internalize an exogenous
ligand, we have obtained CHO and A9 cells (both of which are not
so transducible by AdV) that express the HA-tagged human P2Y2 receptor
(HA tag on the extracelluiar N-terminus) by retroviral gene transfer. HA-
P2Y2 receptor expressing A9 cells, but not control cells, stain with
fiuorescently labeled anti-HA Abs under resting conditions. With agonist
CA 02295315 1999-12-15 gIMB_A5-~
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(ATPrs (1 ~ Mu axpaauro, approxinn~ly g0~6 of tho roooptors are
ir~brnr~llssd wnhln 45 minutes. Ths IntemalJzadon of PZY~ reoepmr is
madlatad vla aortod pits.
Next, a bi-opeoHto antibody (bs~) tpproe~oh v~g used tv east
whethor P2Y~ raclp~or could modlata 0~ne transtor. Ardlbody
HA.11 (BabtO) aps~lns~t ~fluerura hsmaaplutln (snti.HA) is dt~obed a~alnst
the HA-Apltvpe innrtsd into en extracellular domain of the human P~Yx
receptor vrfilch b oxpnavd In 18Z1N1 human estrocyboma astls. The
brid0lng antibody is produ0ld by rstCdng an ptllf-flbsr (knob) antibody with
rr~MeleknldoDsnt~yl-N-hydn~oqlsuliatuodlnlmld~ s~ (Sutfo-MBS, pierce,
Rodcford, It.) or N-(~alalmidobutyryloxY) tulfoauodnihlido e~itsr (sutlb-
GM~8, Pforoe. l~eldvc~, It-) a! neub~f pN. Aftor roduction of anti-HA by
rn,~aptoethylamfna, tnd deealttnp. the two evtlbodfes are mixed.
enablltt~ disuttde Ct~ost-link folmstion, Bt-functional antibody is putMod by
soqua~lal chromttopraphy aver fiber pn~toln and HA ootumns.
Ustrt~ thl! brAbs Itdainst the Ad Itbor (itnob) and the HA oplLvpo
tai (alqbar x aliA), blndlr~ of btrAb and AdV to HA-PxY~ ncoptor, but not
to null eocprsatnp A9 ceth has been dsmonstrsted. AdV bound to A9,HA-
P2Ys oelte tn the pressrtoo, but not fhs aber~ttos, of bl~Ab.
Mdre Impo~tMly, PZYg rovaptor spaaflc Oohs transfer in AS and
CHO oslh has bostt tchieved ulinp tho bt~Ab approach. In one protaool
omp~Yl~~ (1 ) ~q~$I eapoeura at a~C of HA P2Y~ racoptor
exp~tlnQ A9.vsnus null vecttir~xpr~bnit~ Ag c:allt to varyiap
oonoentrsrtioni of bs~ldt (anb-HAltntl-fiber knob; aroduaed by Dr. R.
Piddes in the lsbotl~bory of Dr. D. 8~t ~ttt~e NtH) i~ollow4d by IIdV~IaoZ
(psrtldes 01~1 d~ (PlcWss, R. J,. eat al.,1908. Hurruen Gene. Therapy y
921 ~Q31 ): (2) Inwballon fbr 1 hour at 3TC with bpanist (A~TF~S.10'~ M),
f~dla0~wod by Incubatbn fbr 24 hour in madlwm: and (3) quandtatton of Qane
tr~nsfsr afAolency by oountlng tnd oalculatmp the percent IacZ potttJvo
oolis, tt was oburved that HA- P~Yz reoaptor oxpnstinp A9 aolls aro
tranrdeosd by I~d-tacZ e~ s function a!ttho conosntratton of b~mAb.
wltarrae nuU A6 cells sro not (Fi~uro 1A). Indeed, It appsan that noariy
1 On9i pena transhr attlciericy is approacttod wlih 30 wylrnl of bs-Abe.
~'1 - ..
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'!n a second protocat using preformed conjugates, aup~~Mat~on of
gene tremaller thAt peaked et ~90 pplml b~Ab waa observed (Flouro ~A).
The fail In tranaduction efNclency at higher bs-Ab vonCentr~ora may
n~iect oompetiteon by unbound be.Ab ibr the tar~e~t. .
similar lncrw~ess tn Qene transfer to HA. P2YrR A9 cells with be.
Aba against h!A snd biotin (aHAJabiobn~ and blotinylstsd ad.rtovirat
we:Evre, Finally, the tpeolnolty of the pane trend~r observed with bs~Ab in
Ag~HArP2Ya-R cells we evaluated (Plaure 48). HA- P2Ya~A9 cstl~
exposed to Ad-L~acZ anQ A"i'Pr5 =howsd almost 140'6 exprwsivn oZ t,,acz.
I~ ~t~tet~ almost no Lack sapnaaion was observed with an Irreiewnt
bs-Ab (ltltt!-ftd9nti,AD P1 ~Itoply~ nor In the prosenol 0f a 40X sx~deas of
!?ree anti-HA sraabody, nor In calls that had been Pre-exposed ibr 24 hour
to a high oonCentnt~tvn oispvnist hereby inducfnp c~tl.~~ ~~p~r
down,reQuhrtton~, 'These data eslsblish thst the NA~P2Yz recep~r
medtstea gene tranei~ir v1a epsClllo In:~tior~s with artd-HA bs.Ab:.
BXAMPIE ~
Exploitation of Other CeilulAr Uplake
Msahanlame to increase AdV~enty Into YYD Ct~lturee
The e~lfsc~venera of pens transfer by tarpe~n~ AdV ~p recepEora
that undergo tntenwll~ion la dope~ent on the irrtemalliration efid~cy
o! the receptor. To lest tt~e inbemaltzotion e!!ldenoy of P2Ys-reoaptcrs, a
hurnrn P2Yrroceptor (~ri an tr~f(cisnsa hentaQlubfn (HA) oplEopv-tag
inserted inbo the extreu~nuler damatn) hay bevn over~cpreaaod In 1~2i N1
human astrooytapma ostt:, S. aromek srtd T, K. Harden, Molsauisr
Phannecotopy 64, d4:4B5 (t sae). P2Y~-HA expreatinp cells were
tnc:ubstsd st 3T°C v~th a non-hydroiyzabte ATP ana>aAua, ATPy9. At
specMc llrne ,point:, after apvn~t ~tddluon, the cells were ~~ wkt,aut
penneablllaatlon In 4~6 petactormaldahyde and west. Monoclonal anti.
HA arnibody wee inarbat~ed with the cells ft~lowe~ by fncubedon with Cy3.
~nlugarid goat pnli~mouee tQ0 secondary aMrbody (Jackson tmrnuno
~s~rch Labs). 1'he eva0ablltty of P2Y~HA receptors to the and-HA wsa
vfeuaitred with a tlaoheosnt m~cro~roope. The aatrooyboma calls wore
1i-t~ . ~$, V
.. .~.,-. __t~ ',~gGRI' 'dN S 9 S 8 R tIItRC:R RRR1 'r7 'Nnr
CA 02295315 1999-12-15
WO 99/00511 PCT/US98/13336
29
fixed and immunostained for the presence of P2Y2-HA-receptor in the
absence of ATPpS and after 30 min ATPyS exposure. In the continued
presence of agonist there is a clear loss of immunoreactivity from the
plasma membrane and the punctate fluorescent signals, indicating
sequestration into endosomes. With ELISA assays internalization was
quantitated to occur with an efficiency of 80% (loss of receptor sites with
ATPyS exposures of 1 hr, Dr. Ken Harden, personal communication). This
indicates that in the astrocytoma cell-line, internalization of activated
receptors occurs efficiently.
EXAMPLE 6
Gene Transfer Across the Apical membrane of Polarized Epithelium
Confluent MDCK renal cells were used as a model of a polarized,
AdV-resistant epithelium. In MDCK cells expressing the HA-P2Y2
receptor, the receptors were localized on the apical surfaces. Cells were
exposed to anti-HA and anti-mouse IgG FITC at 4°C, and receptor
localization determined by confocal microscopy. It was observed that HA-
P2Y2 receptors in the apical membrane of polarized MDCK cells
desensitizes, in part, by intemaiization of receptors. Bs-Abs directed to
2o HA- P2Y2 receptors sequentially administered with AdV-GFP (green
fluorescent protein) and ATPyS transduce HA- P2Y2 receptor, as
compared with Neo-expressing MDCK cells. Thus HA-P2Y2 receptor
specific gene transfer has been achieved in polarized, AdV-resistant cells.
EXAMPLE 7
Gene Transfer in A9 Cells
Expressing the Bradykinin II (B2K) Receptor and
in CHO Cells Expressing the P2Y2 or (i2 Receptors
The general applicability of the above approach has been
3o demonstrated in other cell types and with other receptors.
A9 cells expressing the HA-epitope tagged BK" receptor and A9
cells expressing neomycin alone were exposed to bi-specific antibodies
(anti-fibre-knob x anti-HA) and AdV in the absence and presence of
SENT BY:SIMBAS ~ ~_25_pu ~0~2 j~rms~ 1999-i2-is 5IM8'S~ f~ ~ ~ ~~ i ~ ~ r$ ~~3
' ~~ 1~ J UN X999
bratdykinln. Briafly, aelle st 4°C were Incubated In the ibrenoe or
proaenos of bfipr~c antibody (ba~Ab, l0irglmt fbr 2 hrs), washed and
exposed bo AdVLacZ (1 t1s° pselides far 2 hra?, weshad and exposed to
bradytdnln (SK,1 yIM for 2 hn at 3?°C). The celte were Irian
mairtteined at
37°C ford hrs undl grne vxpneslon woo asese~svd by standard
teehnlques. rll~4ln 3 shows that a~fllr~ant qenv expression occurs only in
HA82k-expraasit~p cell: Ineub~d with botfi bs-Ab and AdV with
enhaneemvnt of gen* transior by activa~irt~ the naovptar with aaonlat.
Alrrb~tt no vxpreelJon was abr~nrod with AdV alone, and only modest
levete ware obsenhd in the prosenoa otAdV t bf-Ab (indivating low-Ivval
rsosptcrtum-ovrr~ even fn the abasncv of i'~pand). Only negllpiblv t.~Z
vxpnee~afon was obsena~ in null A9 cells re~t~dles~ of tTertrr~arit.
In additional studies, CHO oeiis (Chinese Wamatsr Ovary cello whk~1 iedt
the AdV atlachmertt ~soapbor) Iftpreaslng 1hv HAwpltopo tapqvd ~-
adretlansvaptor afld wRd~yPa (Vltt) CMO cells wets ~raquvr~tially expoa~! ~
InasaelnQ oonosnthtlons o~( bf-l~sdfiC antibodloa (~ttKl-fibt~rta~ob x ardl-
HA) and AdV and flnaAy ~ isaprv~ronot. 9~ie~lly, ~s st 4°G were
Inarbabad tn the absanve or prssvnoa o! b?'speclflc antibody (bi-Ab, f?,1-
l0~lml for Z hn), wevshad and vxposad to Adll~cx (10'° pattkiea for 2
hn), washed and exposed to i=opraterenol (iONM for 2 nrs at 3T°C). The
mile ate then metr~talnod at 37°C for 24 hr~ until gvrw vxprasaion wAa
anoaed by denalbometry of L»C.~ expressing t;sslla shown as arbitrary
unite. RIgWrv dA~l~ ahvwa ba-Ab dode~deprndsnt tncrorsaa in bane
expresaJon only In CHO orll: vxpreseinq HA.PZYrR ~l~uro ~A) cr HArpz
{Fl~un sA) rec~ori.
~XAI~APLE g
Biotteylfatad ~l~oniale
Mother appr~Owd~ tv taroet vactan lei the P2Y~ roaeptpr (or any
ether 7-TM raoepto~ 11 by ch8rriical linkage to s tnddifled
apontatlantaponiat mole0uls es s tarpvt molecule. A prot4~rpi e~ponht-
iir~kvr la bbbn (B~.UTp, which oor>roatns a 16 carbon linker oonneofina the 6
posittlon o~f the pyrimldlne bees to biotin, 8-UTP is an a9onlot of PAY=
_ . . so
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SENT BY:SIMBAS ~ CA 0229s31s 1999-i2-is gIMBA~~ 819 953 ~5r38~14
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ors (Flpure r). Flu4teeoones studtea have dl~montb~abd that when
CHO oetts exptssslnp P2Y,~n~ptoro aro sxpOeed to B~UTP ooryupsted to
strs~vidin Texss Rid (TR), the 9-IJTP trippehs and is i~ernaliaed wttt~
the PZYa tscep~ore. .
In a RWhar study, pens tar~cttnp by 8-UTP was evaluated In Aq
tills sxprw~sinp 1~IA~P2Ys r~sceptvrs. HA~P~lfs-AD cells were sequentially
aucposed to BrUTP, stnptavldln (SA). and B~AD (blaMn labetsd adenovirus
a~cprssetnp LacZ). Approximately, ~0% of NA~P~Y~ cells
demonstrated LecZ expASllon (>f:lyure 8). In corstr's~t, only Iow levels of
~saZ sxpreesion were obsdrwd in wtld-type A9 ostls, ,
i
~7UMPLp p
Dlnuc!~atlde A~ontetr
Non-hydroly:~bie bidoplcs4y aatlvr enrlops of UTP 1br Ilnicape to
vectors have been developed. 'fhe dinucl4oNdo UePi his idea! properdes.
As shown In l~t~uro 8. U~P~Is equ'~pvro~tWith UTp in IhdudnQ a btolvplcal
refponse (inoMlfol Phosphate csleate) end is ra~rtstar~ to hydrolysis In
aysbc fibroNs (CP) sputum.
. pXAMPLE :10
Inwstlpetlon Into tho AdV-Inboras~bon
processes tn Humen PD wnd IIYD Gulturos
The aw~3ee l~0dns aro ~epor~d to rnvdlaf~s i~emall:~tion but not
ettechmant o1 Wd lr~bo splthelio) cells !n vitro. Tha foc~lizotivn of the ~ .
membrane-bvurut acv~~ tnboprtns to the aplasl endl0r bt~e~el
mvrnbrsvea of WD wlturss Is aruClal In ur~dsrnta~tdlt'~ the rolaa of these
molevules in AdV~mvdisted asps trsnaibr. The avsliabfllfy et a number of
different ar~lbodies to these Inteprino ello~ws their lochttan in polar~xed
sp>thslla~ ~o be detemnlned. The Inteptfi~ ere also teospton for pep~des
oontainlnp R~3D amino add sequences. A number o! studlrs havo shown
thst RGD~pspt~s inhibit AdV-mediated gene trsnsf~ tv epnhellal cells by
irrbsraction with the acv~aa inte~rlns. This i"rxample mustrates the efl~ct~r
of
__ .......... , _ .. 31
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ROD piptldee cn AdV-binding. t~rmxilzador~ and trtnoQsne axprebeton in
PD ~tt~d WD cuiatr~ls.
An tnteprfn u~tibody~spr~ia lmmurropr~ecipttation procedure has
been developed tnltlally with RTE WD caftans to Iocallze the ay~iye .
ir~beprin;. 8inos tJ~ttbodia to these Intprfna are not admmsrc>olly
available, ws hws obtained en antibody (R93a. a kind Qitt irvm Dr. 8~von
Atbelda. Unfv. of Penn, PA). reined a~pafnst the human endothelial ref!
vttronectln receptdor whloh has barn lbu red to cross-reed with rst aw~a
U~teptine is used, BrieMy. etwar the apical or basotatetat dor~tns of NVD
cultures were exposed to 9uito-NH8-9iottn (0.3 mglmt, Pteroe) et ~1°C
to
blOtinytabe only sxfernat membrane pro~talM, After soiubttizatlon of the
oelle In a nondsnaturfnp iysls buffer (in th~ preasnoe o! protease
~hlbltrari). proteins we~a Immunaproctpftabsd and ssperoted by Western
analysis on a 4~1~% avryiamtda gel (Noveut) under non.~edudng
condidona. The bloanytatsd pro%tno wvro probed with rUepdwtdin-
co~r~ugatod pdroxidaH eooondary antibody and detecaed by JCL analysis
(Supersbnai C~.~f~IRP, Pivrct). Tho btolMylat4d protslru idsnti~wd by
Rd38 ore shown in ft~urv 6 (bands at l~SkD oorrospond bo the av and
97kD sr~e p~ ~eubunitl~nd appear fio bo pfdserst in the basolatat'tl
membranes of RTE oelis and 1n hlvLa oetis but absent from the apical
mernbtatne ofithe WD cutiuns, w0gsssinp that rat vtbonectfn receptors
may not be fooatsd apicalty In these oul~rs-tY~ss. 'rhe at»encs of these
,.
intsprlns M tht sp'~oa1 mare~bnns oouid eaoount fior the low rato of AdV-
lntrsmatlzation Into these vultures. ~ ,
eXAMPL.~ i ~
IdrntMcatton and Locaftxation of
inleprlna present on Human PD and 1ND Cuttuns
Protsjne from firs sptcal andlor bae~iaberal membranes of human
Pp and WD cutbrros Art ssledtvety tsototed by exposing Mdlvldusl
surietces to Sulpho~NH8~Btotln at ~°C. Standard inlmunopreotpitatlons
experiment srs ps~rmed with seltvdve human errllbcdlee (LM808, uv~°
P1 Fo, wvps i Vt~R'f~f7, ay ~ P4G11; pt : C06~. ~~ i anti-~6 and RB3S,
!Z ' "''
9 q ~ RYOt ~ 6 686 t vZ vflr
... ._~a 'a-_e5sl aH
CA 02295315 1999-12-15
WO 99100511 PCTNS98/13336
33
av~33i5 ; all obtained from Chemicon Inc, CA.). This procedure allows for
detection and localization of the av~3 integrins to the apical and/or
basolateral membrane.
EXAMPLE 12
Interference of AdV-Internalization by RGD Peptides.
Adenoviral attachment, internalization and transgene expression in
PD and WD cultures will be measured, as described above, in the
absence and presence of RGD-peptides. Hexa-peptides, the bioactive
o GRGDSP (Gibco-BRL) and the inactive control peptide GRGESP (Gibco-
BRL) will be administered to PD and WD cultures at a final concentration
of 0.1-4.0 mg/ml for 2 hrs at 4°C before the addition of AdV
(10'° p/ml).
Cyclical RGD peptides (Immunodynamics, La Jolla, CA.), reported to be
more potent at reducing AdV-mediated gene transfer, are also used.
~5 Analyses of AdV-attachment, internalization and transgene expression are
performed as described above.
EXAMPLE 13
Investigation of the Cellular Uptafce Processes for AdV-entry
2o Initial attachment of AdV to epithelial cells occurs via the fiber
(knob) protein. It is unclear whether fiber protein alone is sufficient to
trigger internalization and endosome formation or whether the role of fiber
is to aid the virus to locate and exploit an inherent endocytotic event.
Internalization of AdV into the cytoplasm however, is mediated, in part, by
25 av~i3~5 integrins. T. J. Wickham et al., Cell 73, 309-319 (1993). It has
been
speculated that av~i~,5 integrins are absent or low in number in the apical
membranes of both WD cultures and cartilaginous airway epithelium (M. J.
Goldman et al, J. Virol. 69, 5951-5958 (1995)), possibly resulting in both
the low rate of internalization and gene transfer efficiency in these cell-
ao types. Therefore, the potential cellular uptake processes that may be
responsible for entry of AdV into cells are investigated. First, to
understand the functional role of the fiber (knob) protein-cell interaction,
we will conjugate knob protein to fluorescent microspheres of the same
CA 02295315 1999-12-15
WO 99/00511 PCT/US98/13336
34
diameter as Ad and assess the initial interactions of the knob-spheres on
PD and WD cells by confocal microscopy. We will specifically test the
hypothesis that increased specific or non-specific binding can increase
internalization and possibly expression. Second, we will overexpress
human av~35 integrin in WD cultures to direct this protein to the apical
membrane. If this is feasible, we will test the hypothesis that av~35
expression on the apical membrane is the rate-limiting step for
internalization and hence gene expression. Third, in order to understand
the cell-entry pathways utilized by Ad, we will investigate cell-lines that
are
either deficient or competent at coated pit receptor-mediated endocytosis.
We will test the hypothesis that high concentrations of AdV may use non-
specific entry pathways to gain access into cells. Finally, a strategy will be
tested, as an experimental concept, to increase the internalization
efficiency of AdV into WD cells by exploitation of other cellular uptake
s mechanisms, i.e., targeting AdV to specific receptor types that undergo
endocytosis when stimulated by exogenous iigands.
EXAMPLE 14
AdV Internalization into HeLa Cell-Lines
2o with Competent and Defective Receptor-Mediated Endocytosis
Adenoviral entry into cells may reflect uptake by a number of
cellular pathways i.e., receptor-mediated endocytosis via coated pits, non-
specific pinocytosis, or phagocytosis. R. M. Steinman et al., J. Cell Biol.
96, 1-27. We will study the role of coated-pit receptor-mediated
2s endocytosis and non-specific pinocytosis on AdV-entry into cell-lines which
have either competent or defective receptor-mediated endocytosis. HeLa
cell mutants have been produced which can overexpress either wild-type
dynamin protein or a mutant form, mDyn (controlled by a TET-inducible
promoter). H. Damke et al., J. Cetl Biol. 127, 915-934. Normally, dynamin
3o is responsible for coated pit endosome formation and functions by
'pinching' off invaginations in the plasma membrane. HeLa cells
overexpressing wild-type dynamin show no functional or morphological
alteration of uptake processes compared to parent cells. Cells
CA 02295315 1999-12-15
WO 99/00511 PCT/US98/13336
overexpressing mDyn form coated pits and invaginate the plasma
membrane but fail to bud coated vesicles into the cytoplasm. Ligands for
receptor-mediated endocytosis (EGF and transferrin) fait to be internalized
into cells expressing mDyn, but ligand-receptor binding, coated pit
s assembly, recruitment of receptors into coated pits and invagination of the
plasma membrane are all unaffected. In the absence of receptor-
mediated endocytosis, non-specific pinocytosis initially remains unaltered
but with time is upregulated to compensate for the loss of receptor-
mediated endocytosis.
EXAMPLE 15
Uptake Processes for AdV-entry into HeLa Cells.
We will use HeLa cells either overexpressing wild-type or mDyn (gift
of Sandra Schmid, Scripps Research Institute, La Jolla, CA) to study the
uptake processes that are prevalent for AdV-entry into these cells. A
range of AdV concentrations will be studied to determine if high titre AdV
leads to cell-uptake by non-specific processes. Briefly, monolayers of
mutant HeLa cells, grown on plastic, expressing wild-type or mdyn
dynamin will be exposed to AdSVLacZ (106-10" i.u.lml, corresponding to
2o an MOI-1-105) at 4°C for 2hrs and then transferred, without washing,
to
37°C for time-periods of 0-24hr, at which point the cells will be
washed and
maintained at 37°C for 48 hrs before ~3-gal enzymatic assays are
performed. Differences in the gene expression observed in the two cell-
lines at specific time-points will reflect the participation of receptor-
mediated endocytosis on AdV-entry. In conjunction, we will perform
comparative studies with fluorescent microspheres (with and without
attached knob-protein) to delineate the interaction of these proteins with
specific and non-specific uptake processes.
CA 02295315 1999-12-15
WO 99/00511 PC'T/US98/13336
36
EXAMPLE 16
Exploitation of Other Cellular Uptake
Mechanisms to Increase AdV-entry into WD Cultures
The effectiveness of gene transfer by targeting AdV to receptors
that undergo internalization is dependent on the internalization efficiency
of the receptor. To test the internalization efficiency of P2Y2-receptors, a
human P2Y2-receptor (with an influenza hemagluttin (HA) epitope-tag
inserted into the extracellular domain) has been overexpressed in 1321 N1
human astrocytoma cells. P2Y2-HA expressing cells were incubated at
~0 37°C with a non-hydrolyzable ATP analogue, ATPyS. At specific time
points, after agonist addition, the cells were fixed without pem~eabilization
in 4% paraformaldehyde and washed. Monoclonal anti-HA antibody was
incubated with the cells followed by incubation with Cy3-conjugated goat
anti-mouse IgG secondary antibody (Jackson Immuno Research Labs).
~5 The availability of P2Y2-HA receptors to the anti-HA was visualized with a
fluorescent microscope. Cells were fixed and immunostained for the
presence of P2Y2-HA-receptor either a} in the absence of ATPyS, or b)
after 30 min ATPyS exposure. In the continued presence of agonist there
is a clear loss of immunoreactivity from the plasma membrane and the
2o punctate fluorescent signals, indicating sequestration into endosomes.
With ELISA assays internalization was quantitated to occur with an
efficiency of 80% (loss of receptor sites with ATPyS exposures of 1 hr, Dr.
Ken Harden, personal communication). This indicates that in the
astrocytoma cell-tine, internalization of activated receptors occurs
25 efficiently.
EXAMPLE 17
Analysis of Transgene Expression in Human PD and WD Cultures
PD and WD cultures are exposed to AdSVLacZ by application to
3o either the apical andlor basolateral membranes over a range of viral titres
(106-10" infectious unitslml: corresponding to MOI range of 1-105) with
incremental exposure times (1-24 hrs), to study the effects of
concentration and time on the gene expression obtained. Vectors used in
CA 02295315 1999-12-15
WO 99/00511 PCT/US98/13336
37
this study are produced and titred by the UNC Gene Therapy Core.
Incubations are performed at 37°C and/or 4°C. The former
temperature
allows potential cellular uptake processes to be studied, while the latter
temperature, is a standardized technique for measuring the initial
attachment of ligands to their receptors, in the absence of receptor
recycling and/or internalization. Gene expression is assessed 48 hrs after
initial exposure to AdV by both qualitative and quantitative means (X-gal
histochemistry and standard colourimetric enzyme assays, respectively).
See Pickles, R. J., et al., 1996. Human Gene Therapy7, 921-931.
While the invention has been described in connection with specific
embodiments thereof, it will be understood that the invention is capable of
further modification. This application is intended to encompass any
variations, uses or adaptations of the invention that follow in general, the
principles of the present invention and including such departures from the
present disclosure as come within known , or customary practice within the
art to which the invention pertains, as may be applied to the essential
features set forth in the scope of the scope of the embodiment of the
invention described above.
