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Patent 2133411 Summary

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(12) Patent Application: (11) CA 2133411
(54) English Title: GENE THERAPY USING TARGETED VIRAL VECTORS
(54) French Title: THERAPIE GENIQUE A L'AIDE DE VECTEURS VIRAUX SPECIFIQUES
Status: Dead
Bibliographic Data
(51) International Patent Classification (IPC):
  • C12N 15/86 (2006.01)
  • A61K 48/00 (2006.01)
  • C12N 15/867 (2006.01)
(72) Inventors :
  • YOUNG, ALEXANDER T. (United States of America)
(73) Owners :
  • YOUNG, ALEXANDER T. (United States of America)
(71) Applicants :
  • YOUNG, ALEXANDER T. (United States of America)
(74) Agent: SMART & BIGGAR
(74) Associate agent:
(45) Issued:
(86) PCT Filing Date: 1993-03-31
(87) Open to Public Inspection: 1993-10-14
Examination requested: 2000-02-04
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/US1993/002957
(87) International Publication Number: WO1993/020221
(85) National Entry: 1994-09-30

(30) Application Priority Data:
Application No. Country/Territory Date
862,795 United States of America 1992-04-03
08/040,748 United States of America 1993-03-29

Abstracts

English Abstract

2133411 9320221 PCTABS00027
A general method for delivering genes to specific target cells
in vivo is described. Enveloped viruses are genetically
engineered to infect specific target cells by replacing the cell surface
receptor recognition domain of viral envelope proteins with
ligands that direct the binding and fusion of these viruses to
specific cell surface molecules.


Claims

Note: Claims are shown in the official language in which they were submitted.


WO 93/20221 PCT/US93/02957

- 41 -
What is claimed is:
Claims
1. A method for expressing a nucleic acid
of interest in a heterologous host cell, said method
comprising
(a) providing a virus whose genome comprises
(i) said nucleic acid of interest and (ii) a hybrid
envelope gene, said hybrid gene encoding an envelope
fragment joined to a targeting ligand, whereby said
envelope fragment does not facilitate recognition or
binding of its normal host cell but which does facilitate
efficient incorporation of said virus into a mature viral
particle and whereby said targeting ligand facilitates
targeting and binding of said mature viral particle to
the surface of said heterologous host cell, and
(b) administering said virus so as to permit
viral infection of said heterologous host cell.

2. The method of claim 1, wherein said
virus is an envelope virus.

3. The method of claim 2, wherein said
envelope virus is a Herpesviridae.

4. The method of claim 2, wherein said
envelope virus is a Retroviridae.

5. The method of claim 4, wherein said
Retroviridae is a Moloney murine leukemia virus.

6. The method of claim 1, wherein said
nucleic acid of interest is DNA.

7. The method of claim 1, wherein said
nucleic acid of interest is RNA.

WO 93/20221 PCT/US93/02957

- 42 -
8. The method of claim 1, wherein said
heterologous host cell is infectious.

9. The method of claim 1, wherein a portion
of said hybrid envelope fragment consists of a receptor
binding domain, an oligomerization domain, a
transmembrane domain, a virus budding domain, sorting
signals, and a signal sequence.

10. The method of claim 9, wherein said
envelope fragment further consists of a fusion domain.

11. The method of claim 1, wherein the
fusion activity of said envelope fragment is performed by
a second protein.

12. The method of claim 1, wherein said
administration is by implanting a container enclosing
said virus into a patient.

13. The method of claim 12, wherein said
virus is inside a packaging cell.

14. A virus, the genome of which encodes a
hybrid envelope protein, said hybrid protein comprising
an envelope fragment joined in frame to a targeting
ligand, whereby said envelope fragment does not
facilitate recognition or binding of its normal host cell
but which does facilitate efficient incorporation of said
hybrid envelope protein into a mature viral particle and
whereby said non-viral protein facilitates targeting and
binding of said mature viral particle to the surface of a
cell not normally infected by said virus.

WO 93/20221 PCT/US93/02957

- 43 -
15. The virus of claim 14, wherein said
virus is an envelope virus.

16. The virus of claim 15, wherein said
envelope virus is a Herpesviridae.

17. The virus of claim 15, wherein said
envelope virus is a Retroviridae.

18. The virus of claim 17, wherein said
Retroviridae is a Moloney murine leukemia virus.

19. The virus of claim 14, wherein said
nucleic acid of interest is DNA.

20. The virus of claim 14, wherein said
nucleic acid of interest is RNA.

21. The virus of claim 14, wherein said
heterologous host cell is infectious.

22. The virus of claim 14, wherein a portion
of said hybrid envelope protein consists of a receptor
binding domain, an oligomerization domain, a
transmembrane domain, a virus budding domain, sorting
signals, and a signal sequence.

23. The virus of claim 22, wherein said
envelope fragment further consists of a fusion domain.

24. The virus of claim 14, wherein the
fusion activity of said envelope fragment is performed by
a second protein.

WO 93/20221 PCT/US93/02957


- 44 -

25. The virus of claim 14, wherein said
administration is by implanting a container enclosing
said virus into a patient.

26. The virus of claim 25, wherein said
virus is inside a packaging cell.

27. A method for delivering a nucleic acid
of interest to a heterologous host cell, said method
comprising
a) providing a virus whose genome comprises
(i) said nucleic acid of interest and (ii) a hybrid
envelope gene, said hybrid gene encoding an envelope
fragment joined to a targeting ligand, whereby said
envelope fragment does not facilitate recognition or
binding to its normal host cell but does facilitate
efficient incorporation of said virus into a mature viral
particle and whereby said targeting ligand facilitates
targeting and binding of said mature viral particle to
the surface of said heterologous host cell, and
b) administering said virus so as to permit
viral infection of said cell.

28. The method of claim 27, wherein said
virus is an envelope virus.

29. The method of claim 28, wherein said
envelope virus is a Herpesviridae.

30. The method of claim 28, wherein said
envelope virus is a Retroviridae.

31. The method of claim 30, wherein said
Retroviridae is a Moloney murine leukemia virus.


WO 93/20221 PCT/US93/02957

- 45 -
32. The method of claim 27, wherein said
nucleic acid of interest is DNA.

33. The method of claim 27, wherein said
nucleic acid of interest is an RNA.

34. The method of claim 27, wherein said
heterologous host cell is infectious.

35. The method of claim 27, wherein a
portion of said hybrid envelope fragment consists of a
receptor binding domain, an oligomerization domain, a
transmembrane domain, a virus budding domain, sorting
signals, and a signal sequence.

36. The method of claim 35, wherein said
envelope fragment further consists of a fusion domain.

37. The method of claim 27, wherein said the
fusion activity of said envelope fragment is performed by
a second protein.

38. The method of claim 27, wherein said
administration is by implanting a container enclosing
said virus into a patient.

39. The method of claim 38, wherein said
virus is inside a packaging cell.

Description

Note: Descriptions are shown in the official language in which they were submitted.



WO93/20221 . 913 3 g 11 PCT/US93/02957

J'~ . .

GENE THERAPY USTNG TARGETED VIRAL VECTORS
Backaround of the Invention
The invention relates to gene therapy methods.
Gene therapy is an approach to treating a broad
range of diseases by delivering therapeutic genes
directly into the human body. Diseases that can
potentially be cured by gene therapy include 1) diseases
associated with the aging population such as cancer,
10 heart disease, Alzheimer' 8 disease, high blood pressure,
atherosclerosis and arthritis; 2) viral infectious
diseases such as acquired immune deficiency syndrome
(AIDS) and herpes; and 3) inherited diseases such as
diabetes, hemophilia, cystic fibrosis, and muscular
15 dystrophy.
Current methods of delivery of new genetic
information into cells in vitro include cell fusion,
chromosome-mediated insertion, microcell-mediated gene
transfer, liposome DNA carriers, spheroplast fusion, DNA-
20 mediated gene transfer, microinjection, infection withrecombinant RNA viruses, and infection with recombinant
DNA viruses (Martin, J.C., 1984, Mol. C~ll Biochem. 59:3-
10). These techniques are not generally applicable,
howevsr, for use in animals or humans because of low
25 efficiency, instability of introduced genes, introduction
of extraneous or undesirable genetic information, and
lack of target specificity.
In one particular example, a favored approach for
human gene therapy involves the transplantation of
30 genetically-altQred cells into patients (Rosenberg, et
al., 1988, New Eng J Med~cine 323:570-578). This
approach requires the surgical removal of cells from each
patient to isolate target cells from nontarget cells.
Genes are introduced into these cells via viral vectors
35 or other maans, followed by transplantation of the

W093/20221 ~13 3 ~1 1 i i; . ' .~ PCT/US93/02g57


genetically-altered cells back into the patient.
Although this approach is useful for purposes such as
enzyme replacement therapy (for example, for
transplantation into a patient of cells that secrete a
5 hormone that diseased cells can no longer secrete),
transplantation strategies are less likely to be suitable
for treating diseases such as cystic fibrosis or cancer,
where the diseased cells themselves must be corrected.
Other problems commonly encountered with this approach
lO include technical problems, including inefficient
transduction of stem cells, low expression of the
transgene, and growth of cells in tissue culture which
may select for cells that are predisposed to cancer.
Finally, inappropriate expression of transplanted genes
~5 in nontarget oells may actually be harmful to patients.
An alternative approach to gene therapy involves
the direct delivery of genes to target tissue in situ.
Two methods for in s~tu delivery of genes have been
developed: biolistic transfer and double balloon
20 catheterization. Biolistic transfer of genes involves
shooting DNA-~oated platinum or gold micropro~ectiles
directly into target tissue. Biolistic transfer has been
successful in the transient expression of genes in the
ear, skin and surgically-exposed liver of live mice
~Johnston, S.A., l990, Natur~ ~46:776-777; Williams,
R.S., et al., l99l, Proc Na~l Acad Sci USA 88:2726-2730).
Double balloon catheterization transduces genes into
cells within a defined arterial wall segment. In this
approach a double balloon catheter is inserted into an
30 artery until the end of the catheter is located within
the target area. Inflation of two balloons at the end of
the catheter creates an enclosed space into which
retrovirus or DNA-loaded liposomes are infused. This
~ method has been successful in the transient expression of
`~ 35 ~-galactosidase genes within a defined segment of the

wo g3,2022. 2 1 3 3 4 1 1 ; ~ , PCT/US93/02gS7


ileofemoral artery of pigs (Nabel E.G., et al., 19~0,
Science 249:1285-1288). Both biolistic transfer and
double balloon catheterization however, althouqh locally
specific, may be nonspecific in the individual cells that
5 they transduce within the target area, creating a problem
of inappropriate gene regulation if the transgene is
expressed in nontarget cells. Moreover, neither
biolistic transfer nor double balloon catheterization
have been shown to be effective for the treatment of
- 10 tissue occupying large volumes such as lungs, muscles,
tumors, or cells of the systemic circulation since the
majority of the cells would be inaccessible for in situ
gene transfer.
A third approach to gene therapy is the delivery
15 of genes to cells in vivo. This approach involves the
introduction of viral vectors directly into patients by
in~ection, spray or other means. Different species of
viruses are engineered to deliver genes to the cells that
the viruses normally infect. Adenovirus, for example,
20 which normally infects lung cells, has been developed as
a vector to target genes to lung cells (Rosen~ield, et
al., 1992, Cell 68 143-155). Most viral vectors,
however, are single purpose vectors since they can only
deliver genes to certain cells. Because the target cell
25 specificity of viral vectors i8 re~tricted to the normal
tropisms of the viruses, viral vectors are generally
limited in that they either infect too broad a range of
cell types, or they do not infect certain types of cells
at all.
Liposomes have been designed to deliver genes or
drugs to specific target cells ~n v~vo. By chemically
con~ugating antibodies or ligands to liposomes, liposomes
have been targeted to specific cells. With this method,
antisense env RNA has been delivered to human
35 immunodeficiency virus (HIV)-infected lymphocytes using

W093/20221 ~ 1334 ~ PCT/US93/02ff~


anti-CD3-conjugated liposomes (Renneisen, K ., et al.,
1990, J Biol Chem 265:16337-16342); chloramphenicol
transacetylase (CAT) genes have been delivered to H2Kk
positive lymphomas in H2Kk-negative nude mice using anti-
5 H2Kk-conjugated liposomes (Wang, C. et al., 1987, Proc
Natl Acad Sci USA 84:7851-7855); and xanthine guanine
phosphoribosyltransferase (XGPRT) genes have been
delivered to immunoglobulin-coated cells using
~ staphy}ococcus protein A-conjugated liposomes (Machy P.,
;~ 10 et al., 1988, Proc Natl Acad sci USA 85:8027-8031). The
major drawback to this technology can be the expense of
mass producing ligand-con~ugated liposomes.
Wu et al. report a method to target naked DNA to
specific cells. Asialoglycoprotein-DNA complexes are
15 targeted to hepatocytes expressing the asialoglycoprotein
;receptor (Wu G.Y., et al., 1991, Biotherapy 3:87-95) .
Similar to the problem encountered with immunotoxins,
however, this ~trategy generally limits delivery of DNA
to cells expressing receptors that are capable of DNA-
: `~
20 internalization.
Antisense DNA technology is a method ror
inhibiting the expression of specific genes with
complementary DNA (Moffat, 1991, Science ~:510-511).
Although antisense DNA is specific in the genes that it
25 affects, it is nonæpecific in the types of cells that it
gets into. This can create problems ~n vivo because it
is desirable that endogenous genes in normal cells remain
unaffected by antisense DNA (e.g., protooncogenes).
Moreover, the cost of manufacturing and administering
30 antisense DNA may be high because the phosphate moieties
of antisense DNA must be chemically mod~fied to allow
passage through tbe plasma membrane, a process which
entails expensive organic chemistry. Millimolar
concentrations of antisense DNA are required to be
35 effective, posing problems of potential toxicity in vivo.

WO93/20221 ~ 3 34fll~ PCT/US93/02g5


Human gene therapy is therefore limited by.the
available technology for gene delivery. Transplantation
strategies, which require surgery, limit gene therapy to
an expensive service industry for a small number of
5 diseases. Targeting of genes in s~tu through local
transduction is generally not precise enough. Viral
vectors limit the delivery of genes ~n vivo to cells that
the viruses normally infect. Liposome technologies may
be infeasible because of the expense of production.
lO Simple ligand-DNA complexes will not introduce genes into
cells unless the receptors, against which the ligan~s are
direct~d, internalize. Acoordingly, currently available
gene delivery systems impose severe limitations on the
spectrum of diseases that can be treated by gene therapy.
Summary of the Invention
The invention features a method for expressing a
nucleic acid of intere~t in a heterologous host cell.
The method involves providing a virus whose genome
comprises i) the nucleic acid of ~nterest, and ii) a
; 20 hybrid envelope gene. The hybrid gene encodes an
;~ envelope fragment ~oined to a targeting ligand, whereby
the envelope fragment does not facilitate recognition or
binding of its normal host cell but does facilitate
efficient incorporation of the virus into a mature viral
25 particle, and whereby the targeting ligand facilitates
targeting ~nd binding of the mature viral particle to the
surface of the heterologous host cell. The method also
involves administering the virus so as to permit viral
infection of the cell.
By "efficient incorporation", is meant that the
hybrid envelope protein ~g incorporated into a mature
viral particle at least 25% a8 frequently as the
corresponding wild-type envelope protein is incorporated
into a mature viral particle.

,_,,
!
WO93/20221 rcT/usg3/o29s7
~1334~11; `
-- 6

In another aspect the invention features a virus,
the genome of which encodes a hybrid envelope protein,
wherein the hybrid protein comprises an envelope fragment
joined in frame to a targeting ligand, whereby the
s envelope fragment does not facilitate recognition or
binding of its normal host cell but which does facilitate
efficient incorporation of the hybrid envelope protein
into a mature viral particle and whereby the non-viral
protein facilitates targeting and binding of the mature
10 viral particle to the surface of a cell not normally
infected by the virus.
In a third aspect the invention features a method
for~delivering a nucleic acid of interest to a
heterologous host cell. The method involves providing a
15 virus that~comprises i) the nucleic acid of interest, and
~-~ ii) a hybrid envelope gene, the hybrid gene encoding an
envelope fragment joined to a targeting ligand, whereby
the envelope fragment does not facilitate recognition or
binding to its normal host cell but does facilitate
20 efficient incorporation of the virus into a m~ture viral
particle, and whereby the targeting ligand faailitates
targeting and binding of the mature viral particle to the
surface of the heterologous host cell. The method also
involves administering the virus so as to permit viral
25 infection of the cell.
In various preferred embodiments the virus is an
enveloped virus, preferably a Herpesviridae, a
Paramyxoviridae, or a Retroviridae, most preferably a
Moloney murine leukemia virus, or the viru~ may also
30 preferably be a Hepadnaviridae, a Poxviridae, or an
Iridoviridae. Similarly tbe virus may be a Togaviridae,
a Flaviviridae, a Coronaviridae, a Rhabodoviridae, a
Filoviridae, an Orthomyxoviridae, a BunyaviridaQ, or an
Arenaviridae, or any other, yet unclassified, enveloped
35 virus.

~"~

.

W093/20221 ~i 3 3 ~ ; PCT/US93/02gS7

- 7 -
In other various preferred embodiments the nucleic
acid of interest may include, without limitation, an
antisense oncogene; a tumor suppressor gene, e.g., a gene
encoding p53, or a gene encoding retinoblastoma protein
5 Rb; a toxin gene, e.g., a diphtheria toxin gene; or a
gene encoding a cytokine, e.g., a tumor necrosis factor,
or an interferon. The nucleic acid of interest may be
either DNA or RNA, e.g., antisense DNA, or antisense RNA,
or a nucleic acid encoding an antisense RNA. The nucleic
lO acid of interest may also be a gene invoking
intracellular immunity, or a nucleic acid therapeutic for
an inherited disease, e.g., an insulin gene, or a cystic
fibrosis transmembrane regulator gene. A "gene that
invokes intracellular immunity" is a gene that confers a
lS dominant negati~e re~istant phenotype to the cell it is
in.~thereby protecting the cell against an invading
aqent.
The heterologous host cell may be a cell that has
acquired mutations that re~ult in a disea~e ~tate,
20 preferably a cancer cell, e.g., a colon cancer cell. The
` heterologous host cell may be a cell infected ~ith a
second virus, e.g., a human immunodeficiency virus (HIV),
a cell infected with an organism, or an infectious agent
such as a bacterium or parasite. The infectiouæ agent
25 may be either unicellular or multicellular. The
heterologous host cell may also be a cell affected by a
hereditary disease, e.g., a pancreatic beta cell, or a
~` lung cell.
The targeting ligand, in additional various
30 preferred embodiments, may include a protein, preferably
a hormone, or an immunoglobulin, more preferably an anti-
tumor associated antigen-specific immunoglobulin, most
preferably an anti-carcinoembryonic antigen-specific
` immunoglobulin, or an anti-HIVgpl20 antigen-specific
35 immùnoglobulin. The targeting ligand may also be a

WO93/20221 Z 1 3 3 ~ PCT/US93/02957
- 8 -
carbohydrate, or a lipid. The hybrid envelope fragment
may consist of a receptor binding domain, an
oligomerization domain, a transmembrane domain, a virus
budding domain, sorting signals, a signal sequence, and
5 preferably a fusion domain. In some cases the fusion
activity of the envelope fragment may be performed by a
second protein. The second protein would therefore
direct fusion of the virus with the membrane of the
targeted cell.
The mode of admini~tration may include, but is not
limited to, l) direct injection of the purified virus; or
2) implanting a container enclosing the virus into a
patient. When the virus is administered inside a
container, the virus is preferably inside a packaging
~lS cell. A "packaging cell" is a cell that supplies viral
;~proteins necessary for production of viral vectors. By
"container" is meant a virus permeable enclosure
containing virus, or containing packaging cells with
virus therein.
"Normal host cell" as used herein, is a cell type
commonly infected by the naturally occurring ~irus. In
contrast, the term "heterologous host cell" or a
"targeted cell", as used herein, refers to a cell that is'
recognized as a function of the targeting ligand portion
25 of the hybrid envelope protein, but i8 not recognized as
a function of the envelope portion of the hybrid envelope
protein. By "targeting ligand" is meant a molecule that
has binding affinity for a moleculQ on tbe surface of a
desired targeted cell. A "hybrid envelope protein", as
30 used herein, is a protein that includes a portion of a
viral envelope protein ~or a biologically active analog
thereof) covalently linked to a targeting ligand. For
example, by a "hybrid immunoglobulin-env protein" is
meant a portion of an immunoglobulin covalently linked to
35 a portion of an envelope protein. A "hybrid envelope

W O 93/20221 ~ 1 3 3 4 1 1 " , `, PC~r/US93/02957


gene" i~ a nucleic acid that provides genetic
instructions for a hybrid envelope protein. By "hybrid
anti-carcinoembryonic antigen-specific immunoglobulin" is
meant a hybrid immunoglobulin-env protein that
5 specifically binds to a carcinoembryonic antigen.
The term "fragment", as applied to an envelope
protein fragment, includes some but not all of the
envelope protein. A fragment will ordinarily be at least
about about 20 amino acids, typically at least about 30
10 amino acids, usually at least about 40 contiguous amino
acids, preferably at least about 50 amino acids, and most
preferably at least about 60 to 80 or more contiguous
amino acids in length. Fragments of an envelope protein
can be generated by methods known to those skilled in the
15 art (e.g., those described herein~.
A biologically active fragment of a viral anvelope
protein is one that possesses at least one of the
following activities: a) it can bind to a cell membrane
if given the appropriate targeting ligand; b) it can
20 enable fusion with a cell membrane; or c) it can enable
incorporation of proteins into a mature viral-particle.
These three biological activities can be performed by the
same envelope protein fragment, or by two separate
envelope protein fragments. As stated above, the
25 envelope fragments of this invention do not facilitate
recognition or binding of the virus' normal host cell.
This is accomplished by either destroying the activity of
the normal receptor binding region by mutation, or by
physically deleting it. A new recombinant receptor-
30 binding region i8 added in its place. The ability of acandidate fragment to exhibit a biological activity of a
viral envelope protein can be assessed by methods known
to those skilled in the art.
The envelope fragment may include the amino acid
35 sequence of a naturally-ocurring viral envelope or may be

WO93/20221 '~1 3 3 4 1 I PCT/US93/02957

-- 10 --
a biologically-active analog thereo~. The biologLca
activity of an envelope analog is assessed using the
methods described herein for testing envelope fragments
for activity.
S Applicants have provided an efficient and reliable
means for ~pecifically delivering therapeutic genes or
antisense nucleic acids to particular animal, plant or
.
human cell types, or to cells of infectious agents.
Their method facilitates treatments for mutagenically
10 acquired, infectious, or inherited diseases, e.g., by
either 1~ antagonizing the effect of an existing cellular
~; gene; 2) complementing the defect of an existing cellular
gene; 3) destroying the target cells through the
introduction of new genetic material; or 4) changing the
15 phenotype of the target cells through the introduction of
` new g-netic material. To specifically target cells for
delivery, a hybrid envelope protein (e.g., an envelope-
antibody or envelope-ligand hybrid) is utilized which
directs specific interaction with a particular target
20 host cell. The viru~es itself, through its efficient
internalization mechanisms, facilitates efficient uptake
of the therapeutic gene. Such viral vectors are uniquely
~; adapted to deliver genes, RNA, or drugs to cell surface
proteins that do not normally internalize.
Another advantage of this invention is that it
overcomes the problem of gene regulation encountered with
other methods of gene therapy. ~enetically-altered cells
must not only synthesize the gene products at the right
location, at the right time, and in the right amounts,
30 but must also be regul~ted in the ~me manner as the
indigenous tis~ue. That is, the transduced cells must
also have all the proper signal transduction mechanisms
to respond to extracellular signals. This may be a
problem in gene therapy for diabetes, for example, where
35 transplanting fibroblasts with insulin genes can be
' ,

W O 93/20221 ~ -~ 3 3 4 1 I PC~r~US93/02957


ineffective or even harmful. As fibroblasts do not
contain the same receptors and signal transduction
machinery as pancreatic beta cells, the insulin genes may
be expressed differently. Targeting genes to the right
5 cells insures that the genes will be properly regulated.
In an additional aspect of the invention, a
selection scheme is devised for creation of hybrid
envelope protein-containing viruses. This strategy will
be feasible for env proteins fused with immunoglobulins
10 or with any ligand that recognizes specific receptors on
cells.
Other features and advantages of the invention
will be apparent from the following description of the
preferred embodiments thereof, and from the claims.
I5 Detailed Description
The drawings will first briefly be described.
Drawinas
FIG. 1 is a representation of a scheme for
constructing retroviral vector pLNCX*.
FIG. 2 is a representation of a scheme for
constructing plasmid LNCenvpA.
FIG. 3 is a representation of a scheme for
constructing plasmid LNC~nv.
FIG. 4 is a representation of a scheme for
25 constructing plasmid pUC Star-Sig.
FIG. 5 is a representation of a scheme for
constructing plasmid LNC-Sig.
FIG. 6 is a representation of a scheme for
` constructing plasmid LNC-ant~CEA.
FIG. 7 is a representation of plasmids used in the
construction of targeted viruses.
FIG. 8 is a representation of a strategy for
generating targeted retroviruses involving construction
of hybrid immunoglobulin-env genes in vitro.

2~33~1, , j
WO93/20221 ~ ~, PCT/US93/02957

- 12 -
FIG. 9 is a representation of a strategy ~or
generating targeted retroviruses involving generation of
pooled virus constructions.
FIG. lo is a representation of a strategy for
s generating targeted retroviruses involving selection and
characterization of targeted virus.
FIG. 11 is a representation of a plasmid
containing a targeted retroviral vector.
FIG. 12 is a representation of a scheme for
10 constructing plasmid pU~ Star-antiCEA.
FIG. 13 is a representation of an alternative
scheme for constructing plasmid LNC-antiCEA.
What follows is a procedure for the delivery of
genes to target cells using targeted viral vectors. To
15 create and target a virus, the receptor recognition
domain of the viral envelope protein is replaced with a
ligand directed against a specific cell surface receptor.
The hybsid envelope protein is incorporated into the
~ viral envelope during the budding proce~s, producing a
- ~ 20 hybrid virus ~n v~vo. Upon infection of a host, the
~ . ~
hybrid virus specifically recognizes its target cell and
resultant fusion with that cell facilitates
internalization (into the target cell) of viral genes,
including the therapeutic gene(s) which are engineered
25 into the viral genome. Such internalization can be
extremely important; for example, immunotoxins, although
efficient at delivering toxin molecules to target cells,
are often clinically ineffective since the cell surface
molecules to which they are targQted do not ~nternalize,
30 and internalization is re~uired for entry of the toxin
molecules into the cells ~Waldmann, T.A., 1991, Sc~ence
252: 1657-1662). Targeted viruses circumvent this
requirement for receptor internalization since the virus
itself contains the necessary cell fusion machinery
(Gilbert, J.M., et al., 1990, J Virol 64: 5106-5113;

~,

WO93/20221 ~ 3 3 9 1 1 PCT/US93/02957

- 13 -
Roizman, B. et al., 1990, in BN Fields, et al., eds.
Viroloov, Raven Press, Ltd. New York).
General Reauirements for Taraeted Viruses
In general targeted viruses are constructed by
5 replacing the receptor recognition domain of the viral
envelope protein with a ligand directed against a
specific cell surface receptor. The ligand can be,
without limitation, an immunoglobulin (e.g., FAb, dAb,
Fd, or Fc), a hormone, or any other synthetic or natural
10 protein that can direct the binding of the targeted
viruses to a cell surface molecule. The ligand is
biologically incorporated into the viral envelope by
genetic fusion with that portion of the normal viral
envelope protein involved in viral assembly and budding.
15 The envelope portion of the hybrid protein consists of an
envelope fragment (or analog thereof) that is sufficient
to direct efficient incorporation of the envelope hybrid
:`
protein into the viral envelope. Preferably, the
;~ envelope hybrid protein no longer directs an interaction
20 between the virus and ~ts normal host cell.
It has been demonstrated that changing~the
receptor specificity of the envelope protein of a virus
changes the virus's tropism. `For example, vesicular
stomatitis virus (VSV) pseudotypes that have their virus
25 envelope replaced with that of a retrovirus acquire the
ability to infect retrovirus infectable cells (Schnitzer,
T.J., et al., 1977, J Gen Virol 23:449-454; Zavada. J.,
et al., 1~72, J Gen Virol ~:183-191), indicating that
species specific protein-protein interactions between a
30 virus core protein and an envelope protein are not
critical for v~rus fusion and penetration at least in
these cases~ Moreover, previous experiments have also
indicated that virus envelopes can tolerate changes in
~ length or conformation introduced into the envelope
-~ 35 protein, e.g., by a conjugated ligand. For example,


' ;:

WO93t20221 ~ ~ 334t~ ` PCT/US93/02957


Gitman et al. have shown that Sendai virus envelo~es
reconstituted with viral envelope glycoproteins,
chemically cross-linked to anti-erythrocyte antibodies
acquire the ability to bind to erythrocytes that had been
5 stripped of the normal virus receptor. Similarly, Sendai
; virus envelopes reconstituted with envelope proteins,
chemically cross-linked to insulin molecules, were able
to bind to receptor-stripped erythrocytes expressing the
insulin receptor. In both cases, envelope binding but
lO not fusion occurred with the receptor-stripped
erythrocytes. Fusion between the conjugated envelopes
and erythrocytes occurred, however, when the conjugated~
e m elopes were coreconstituted with t~e normal viral
hemagglutinin/neuraminidase and fusion proteins (Gitman,
15 A.G., et al., 1985, Biochem 24:2762-2768).
,
` Preferably, the targeted virus contains l) a viral
envelope derived from a host cellular membrane; 2) a
transmembrane hybrid envelope protein that directs the
binding and penetration of the virus to specific target
20 cells; 3) a transmembrane envelope protein that directs
he fusion of the targeted virus with the cel~ular
membrane of the targeted cell for viral penetration
(e.g., the targeting protein itself or another envelope
protein); 4) viral core proteins; 5) a foreign gene(s) of
25 interest; and 6) all necessary viral and genetic
components for penetration and expression of genes
contained in the viral genome. The transmembrane hybrid
envelope protein consists of l) determinants that enable
the hybrid protein to become processed and incorporated
30 into viral envelopes; 2) det~rminants that enable fus~on
of the viral envelope with the targeted cellular
membrane; these are essential for penetration of the
targeted virus; 3) a ligand determinant that enables the
targeted virus to recognize and bind to specific
35 receptors on target cells. The viral genome may also


~:~

W093/20221 ~ 1 3 3 4 1 1 PCT/US93/02957


include bacterial selectable markers (e.g., ampicillin
resistance) andlor a mammalian cell selectable marker
(e.g., neomycin resistance).
The transmembrane hybrid protein is constructed
S genetically by ~plicing the cell surface receptor binding
domain of a ligand gene to a portion of the viral
envelope protein gene. The transmembrane hybrid protein
must retain those portions of the envelope protein that
direct the effioient post-translational processing,
lO sorting and incorporation of the protein into the viral
envelope.
The following domains must be considered in
constructing the hybrid ligand-envelope protein:
l. The receptor bindina domain
;~ 15 The receptor binding domain is that portion of the
envelope protein that recognizes and binds to cell
~urface receptors. In hybrid envelope proteins, this
; portion of the envelope protein is replaced with ligand
sequences. The receptor binding domain of retrovirus
20 envelope proteins h~s bQen localized to the SU subunit
(Coffin, J.M., l990, in BN Fields, et al., ed~. Virology,
Raven Press, Ltd., New York). Since the SU protein of
retroviruses is coded for 5' to the transmembrane
pro~ein, replacement of the amino-terminal sequences of
25 the envelope protein with ligand sequences poses no
pro~lem for the creation of a functional hybrid ligand-
envelope protein.
2. Proteolytic cleavaae site
The envelope protein of retroviruses is
30 synthesized as a polyprotein which is later
proteolytically cleaved to form SU and TM heterodimers.
In construction of the hybrid ligand-envelope protein,
the proteolytic cleavage site should b~ eliminated. Tbe
proteolytic cleavage site sbould be eliminated either by
3S deletion or by site directed mutagenesis. Perez and

W093/20221 ~3341~` ~ i PCT/US93/02957

- 16 -
Hunter have demonstrated that elimination of the
proteolvtic cleavage site does not block transport or
surface expression of Rous sarcoma virus envelope
proteins (Perez, L.G., et al., 1987, J Virol ~1:1609-
5 1614).
3. Oligomerization domain
The envelope proteins of many animal viruses
associate to form trimers (Fields, B.N., et al., 1991,
Virology, 2nd ed., Raven Press, Ltd., New York).
10 Trimerization of the envelope protein is thought to be
essential for the proper transport and insertion of
envelope proteins into the viral envelope (Singh, J. et
al., l990, Embo J 9:631-639; Kreis, T.E., et al., 1986,
Cell 46:929-937). Therefore it is important that this
15 domain be retained in the hybrid ligand-envelope protein.
The~tri~erization domain likely resides in the
transmembrane TN protein of retroviruses (Einfeld, D., et
al., 1988, Proc Natl Ac~d Sci USA 85:8688-8692); hence,
creation of a functional hybrid ligand-envelope; 2Q rètroviral protein lacking the æu subunit is possible.
Some viral envelope proteins may oligomerize to form
stoichiometric combinations other than trimers.
4. Fusion domain
The fusion domain is a hydrophobic stretch of
25 amino acids that is involved in fusion of the virus
envelope with the cell membrane ~Wiley, D.C., et al.,
1990, in Fields, B.N. et al., eds. Yiroloqy, 2nd ed.,
Raven Press, Ltd., New York). Viral fusion allows entry
of the viral core proteins and genome into the cell. In
30 influenza virus, the fusion domain, located in the amino
terminus of the envelope HA2 protein, is sequestered in
the hemagglutinin trimer until a low pH-induced
conformational change allows presentation of the fusion
domain to the cell membrane. Trimerization of the
; 35 envelope proteins can prevent constitutive expression of
, ~

:^``
wo g3/2022- ~ 1 3 3 4 1 1 rcT/us93/029s7

- 17 -
fusion activity by sequestering it within an internal
hydrophobic pocket. A potential fusion domain has been
located within the extracellular portion of the gp37 TM
protein of Rous sarcoma virus (Hunter E. et al., 1983, J
S Virol 46: 920-936). Similar hydrophobic fusion sequences
have been noted in the plSE protein of Moloney murine
leukemia virus (Mo-MuLV) (Chambers, P., et al.,l990, J
Gen Virol ~1: 3075-3080).
In constructing a hybrid ligand-envelope protein,
10 it may be necessary to eliminate the fusion domain to
pr vent the possibility of constitutive fusion activity,
~; a state that may impair the infectivity of targeted
viru-es~. Therefore two proteins may be incorporated into
the viral envelope of targeting viruses. The first
-15 protein i8 the hybrid ligand-envelope protein which
directs targeting of the virus but lacks fusion activity.
The~s-cond protein is an envelope protein possessing
fusion activity but lacking a receptor binding domain.
This type of situation is observed for paramyxoviruses
20 where one envelope protein is dedicated to targeting
while ~nother carries out fusion (Xingsbury, D.W., 1990,
B.N~ Fields, et al., eds. Yirology, 2nd ed., Raven
Press, Ltd., New York.). Where it is not necessary to
prevent constitutive fusion activity, both activities may
25 be included in one protein.
5. Transmembran~_domain
The transmembrane domain is a stretch of
approximately twenty or more amino acids that anchor the
envelope protein to the viral envelope. It is located
30 within the pl5E protein of Moloney murine leukemia virus
(Chambers, et al., supra). Retention of the
transmembrane domain is thought tQ be essential since
deletion of the transmembrane domain results in secretion
of the synthesized envelope protein (Peres, L.G., et al.,
35 1987, J Virol ~:2981-2988).

W093/20221 ~3~ 4~ PCT/US93/02957

- 18 -
6. Virus buddina domain
Amino acid sequences within the envelope protein
may be involved with the exclusive incorporation of viral
envelope proteins into viral envelopes and with virus
5 budding~ The virus budding domain directs the hybrid
ligand-envelope protein into the viral envelope. These
~equences are thought to reside within the portion of the
envelope protein facing the inside of the virus and may
involve specific protein-protein interactions between
10 envelope proteins and viral core or matrix proteins.
Although Perez Qt al. demonstrated that deletion of the
carboxy-terminal sequences of the Rous sarcoma virus env
~n protein resulted in normal budding of the mutant virus
(Perez, L.G., et al., 1987, J Virol 61:2981-2988),
15 evidence exi~ts that, for other viruses, interactions
between envelope proteins and viral core proteins may
-~ direct virus assembly and envelopment (BN Fields, et al.,
eds., 1990, Viro~Vgy, Raven Press, Ltd., New York).
7. Sortina 8ianal8 and other si~nals
Sorting signals are determinants that direct the
envelope protein to the correct intracellular location
during post-translational processing. These sequences
insure that the envelope protein passes through the
endoplasmic reticulum, Golgi apparatus, and other
25 organelles until it eventually reaches the viral
envelope. Other signals that may have to be retained in
the hybrid ligand-envelope protein are glycosylation
sequences and sequences involved in effective
conformation of the envelope protein (e.g., disulfide
30 bonds).
8. Sional seauence
The signal sequence is an amino-terminal
hydrophobic stretch of amino acids that directs the
envelope protein into the endoplasmic reticulum. The
35 signal sequence, which is later proteolytically cleaved,

W093/20221 ~1 3 3 411 PCT/US93/02957

-- 19 --
is essential for the hybrid ligand-envelope protein to
become located in a membrane.
The diversity of signals and domains that must be
considered in constructing targeted viruses requires that
S precise and correct splicing of ligand and envelope genes
occur. The present invention-describes a selection
scheme for constructing targeted viruses whereby the
ligand gene is spliced to an envelope gene fragment; this
hybrid gene codes for those portions of the envelope
10 protein which are required to direct efficient
incorporation of the resultant hybrid envelope-ligand
protein into the mature viral particle. According to the
selection scheme, cell surface receptor binding domains
of ligand genes are randomly ligated to progressive
15 deletions of viral envelope genes. The correct
co bination of ligand and envelope sequences is
determined by a selection scheme for the production of
bioiogically active targeted virus. The selection scheme
-
not only produces targeted virus but simplifies the
20 construction of future targeted viruses.
~; a,~peoific Exam~l~ of a Taraeted ~etrovirus
There now follows an example of a recombinant
retrovirus which targets and infects particular host
cells for the purpose of delivering to those cells a
25 desired therapeutic gene. This example is provided for
the purpose of illustrating, not limiting, the invention.
Moloney murine leukemia virus (Mo-MuLV) is a mouse
ecotropic retrovirus. A recombinant Mo-NuLV based
retroviral vector that is targeted to colon cancer cells
30 is constructed. The targeted retroviral vector delivers
the neomycin resistance gene to colon cancer cells.
Targeting to human colon cancer cells is accompl~shed by
incorporating into the viral envelope hybrid
i D unoglobulin-env proteins directed against
35 carcinoembryonic antigen. Carcinoembryonic antigen (CEA)

WO93/20221 2 13 3 4 ~ ~ PCT/USg3/02957

- 20 -
is a tumor associated antigen expressed on the surface of
human colon cancer cells but not on the surface of normal
adult cells. The CEA glycoprotein, possessing multiple
membrane spanning alpha helices, does not internalize in
5 response to ligand (Benchimol, S. et al., 1989, Cell
57:327-334). A protein that is homologous to
- carcinoembryonic antigen has recently been shown to be
the receptor for mouse hepatitis virus (Dveksler, G.S.,
et al, 1991, J Virol 65:6881-6891).
For the purpose of this illustration, a single
~ variable region of the heavy chain of anti-CEA is fused
; to a portion of the env gene. Single variable heavy
`~ chain fragments (dAb) have been shown to be as effective
in antigen binding as fragmented antibodies (FAb),
15 oontaining both beavy and light chain fragments, and
intact monoclonal antibodies (Ward, E.S., et al., Nature
544-546). The function of immunoglobulin-env
proteins is not limited, however, to the use of dAb's and
~- can be applied with FAb's, Fv's and mAb's.
20 Modification of the retroviral vector LNCX
: ~ ~ LNCX i8 a Moloney murine leukemia virus based
etroviral vector contained in the plasmid pLNCX (Miller,
A.D., et al., 198~, Biotechniques 7:980-990). pLNCX
contains a unique HindIII and ClaI cloning site for
25 expression of inserted genes, a cytomegalovirus ~CMV)
promoter, a polyadenylation site (pA), retroviral long
terminal repeats (LTR) for retroviral RNA transcription
and reverse transcription, a bacterial neomycin
resistance gene (Neo) which conveys resistance to both
30 neomycin and G418, a bacterial origin of replication
(Or), a bacterial ampicillin resistance gene (Amp), and a
retroviral RNA packaging seguence (~). LNCX is modified
to contain a unigue SalI site as shown in Figure 1.
pLNCX is Iinearized with XbaI and subcloned into the XbaI
35 site of the phagemid BluescriptII SX~ (Stratagene, La

~ " ~

WO93/20221 ~ l 3 3 4 1 1 PCT/US93/02gS7


Jolla, CA). Single stranded DNA is purified and the
unique BstEII site of LNCX is converted into a SalI site
by site directed mutagenesis with the oligonucleotide
5'-GCAGAAGGTCGACCCAACG-3 ' (SEQ ID NO: 1) . The BstEII
5 site is located within the extended packaging signal (~+)
of Mo-NuLV RNA (Bender, M.A., et al., 1987, J V~rol
Çl:1639-1646; Adam, M.A., et al., 1988, J Virol 62:3802-
3806; Armentano, D. et al., 1987, J Virol 61:1647-1650).
Conversion of the BstEII site to SalI does not affect
10 packaging since this region has been determined to be
dispens~ble for efficient packaging (Schwartzberg, P., et
al., 1983, J V~rol 46:538-546; Mann R. et al., 1985, J
Virol 54:401-407; and Mann, R., et al., 1983, Cell
33:153-159). The BstEII site is converted into a SalI
15 site because BstEII sites, but not SalI sites, frequently
occur in heavy chain genes (Chaudhary, V.K., et al, 1990,
Proc Natl Acad Sci USA 87:1066-1070). The SalI
containing plasmid is recircularized with XbaI and DNA
ligase to form the plasmid pLNCX*.
20 Clonina of the Mo-Mu~V env ~rotein in p~NCX*
The Mo-NuLV env gene is cloned into pLNCX* as
shown in Figure 2. The Mo-MuLV env gene is excised from
plasmid p8.2 (Shoemaker, C., et al., 1980, Proc Natl Acad '
Sci USA 77: 3932-3936) as a 1.9kb ScaI-NheI fragment. The
25 l.9kb ScaI-NheI fragment contains the entire coding
region for the pl5E transmembrane protein and the
majority of tha coding region for the gp70 SU protein.
The 5'-protruding ends are digested with Sl-nuclease, and
HindIII linkers (5-CCAAGCTTGG-3'; SEQ ID N0: 2) are
30 added. The env gene is cloned as a HindIII fragment in
the HindIII site of pLNCX* to form plasmid LNCenvpA. The
orient~tion of the HindIII env fragment is such th~t it
can be transcribed and expressed from the cytomegalovirus
~; (CNV) promoter.



:; :

WO93/20221 ~33 ~ PCT/US93/02957

- 22 -
Modification of LNCenvpA to LNCenv
LNCenvpA is cloned as an XbaI fraament in phagemid
pBluescript II SK~ for additional site directed
mutagenesis (Figure 3). The env encoding HindIII
5 fragment contains a polyadenylation signal that may
interfere with the polyadenylation signal provided by the
viral vector. The AAUAAA polyadenylation signal is
therefore changed to AAGAAA by site directed mutagenesis
with the oligonucleotide 5'-GTTTTCTTTTATC-3' (SEQ ID NO:
10 3). The HindIII site located at the 3' end of the env
gene is eliminated by site directed mutagenesis with the
oliqonucleotide 5-CAAGCATGGCTTGCC-3' (SEQ ID NO: 4). The
env containing retroviral vector is recircularized by
XbaI restriction and ligation to form plasmid LNCenv.
15 Molecular clonina of anti-CEA immunoalobulin aenes
cDNA encoding the mature variable region domain of
~ ~ anti-CEA heavy chain genes is cloned as an XhoI-SpeI
;~ fragment using the polymerase chain reaction (PCR) and
RNA template. RNA is derived from the spleen of mice
20 immunized against purified carcinoembryonic antigen.
~::
Alternat~vely, RNA can be derived from hybridoma cell
lines that ~ecrete monoclonal antibodies against CEA,
e.g., 1116NS-3d (American Type Culture Collection
CRL80~9) or CEA 66-E3 (Wagener, C., et al., 1983, J
2S Immunol 130:2308-2315).
The following PCR primers hybridize to cDNA
encoding the aminoterminal end of mature heavy chain
genes (Stratacyte, Inc.). The degenerate primers
introduce an XhoI site which is underlined.

W~93/20221 ~ ~ 3 ~ PCT/US93/02~7

- 23 -
5' AGGTGCAGCTGCTCGAGTCGGG 3' (SEQ ID NO: 5)
5' AGGTGCAACTGCTCGAGTCGGG 3' (SEQ ID NO: 6)
S~ AGGTGCAGCTGCTCGAGTCTGG 3' (SEQ ID NO: 7)
5' AGGTGCAACTGCTCGAGTCTGG 3' (SEQ ID NO: 8~
5' AGGTCCAGCTGCTCGAGTCTGG 3' ~SEQ ID NO: 9)
XhoI

The f~llowing PCR primer hybridizes to
immunoglobulin heavy chain mRNA within the region coding
for the J-region and introduces SpeI and BstEII sites.
5' CTATTAACTAGTGAC~GTTACCGTGGTCCCTTGGCCCCA 3' (SEQ
ID NO: 10)
SpeI BstEII
The amplified anti CEA variable hea~y chain DNA is
cloned as an XhoI-SpeI fragment in an ImmunoZ~P H vector
(Stratacyte, Inc.) (Mullinax, R.I. et al., 1990, Proc
Natl Acad Sci USA 87: 8095-8099). ImmunoZAP H is a
modified lambdaZAP vector that has been modified to
express in E.coli immunoglobulin variable heavy chain
fragments behind a pelB signal sequence. The procadure
20 could similarly be performed by expressing immunoglobulin
variable light chain fragments in a packaging cell line.
Identi~ication of hiah affinitY anti-CEA clones
Clones expressing high affinity anti-CEA
antibodies axe identified by a f ilter binding assay . The
25 anti-CEA phage library i6 screened by nitrocellulose
plaque lifts with ~l25I]bovine serum albumin conjugated to
CEA, as previously described (Huse, W.l)., et al., 1989,
Science 246:12~5-1281). High and intermediate affinity
anti-CEA clones are chosen for further manipulation.
30 Construction of plasmid LNC-immuno~lobulin
Two strategies are presented for creating plasmid
LNC-immunoglobUlin ( in this example, LNC-an~iCEA, which
codes for an anti-CEA immunoglobulin gene). LNC-
immunogl o~ul in vectors encode an immunoglobulin peptide

W O 93/20221 ~1 ~3~ P~r/US93/02957

- 24 -

fused to an amino-terminal signal se~uence. Some amino
acidæ at the amino-terminal end of the mature
immunoglobulin peptide have been modified by the PCR
primers used to generate the immunoZAP library.
5 Moreover, the design of the LNC-antiCEA plasmid results
in the insertion of an extra amino acid at the amino
terminal end. These amino acid changes do not affect
antigen binding because 1) ~he amino acid changes are
conservative; 2) the affected amino acids are normally
10 variable at those sites; and 3) the affected amino acids
occur within the framework region of immunoglobulins`
which has been shown not to participate in antigen
binding or conformation of the antibody (Relchman et al.
1988 Nature 332:323-327). It is for these same reasons
15 that cleavage o* the signal seguence from mature peptide
will not be affected.
~ Both strategies for creating LNC-immunoglobulin
; ~ rely on the use of plasmid pUC Star-Sig, the construction
of which is presented below.
An immunoglobulin signal sequence is cloned into a
modified pUCll9 vector to create pUC Star-s~g.as follows
(Figure 4). pUCll9 is a phagemid containing a polylinker
cloning site. The multiple cloning sites of pUCll9 are
replaced with new restriction sites by insertion of the
25 following polylinker into the HindIII and XbaI sites of
pUCll9.

Hind~I P~tI XhoI BclI SpeI NotI ClaI Xbal
5'-AGCTTCTGCAGGCTCGAGTGATCAACTAGTGCGGCCGCATCGATT-3' (SEQ

ID N0: 11)
30 3'-AGACGTCCGAGCTCACTAGTTGATCACGCCGGCGTAGCTAAGATC-5'(SEQ
ID N0:12)

The modified pUCll9 is called pUC Star-l (Figure 4). The
restriction sites may be further separated by small

W093/20221 ~ 1 3 3 g 11 rCT/US93/02957

- 25 -
linkers, if adjacent restriction siteæ interfere with one
another during digestion.
The signal sequence from an anti-NP immunoglobulin
heavy chain gene is isolated from plasmid pcDFL.l (Ucker,
S D.S., et al., 1985, J Immnol 135:4204-4214) as a -330bp
PstI fragment. The 330bp PstI fragment is subcloned into
pUC Star-l to yield plasmid pUC Star-Sig (Figure 4). The
PstI fragment is oriented so that the signal sequence can
be expressed.
o a. Cons~uç ~ on of L;NC-immunoalobulin through
plasmid LNC-Sig. an immunoalobulin expression vector.
LNCX* is converted into a eukaryotic
immunoglobu}in expression vector (Figures 4 and 5). An
immunoglobulin heavy chain signal sequence and XhoI-SpeI
15 cloning sites are inserted behind the CMV promoter of
plasmid LNCX* to allow expression of the PCR amplified
im~unoglobulin genes. Conversion of LNCX* is as follows.
The immunoglobulin heavy chain signal sequence is
recovered from pUC Star-Sig a8 a HindIII-ClaI restriction
20 fragment ~nd cloned into the HindIII-ClaI sites of LNCX*.
The resulting plasmid, LNC-Sig contains a retroviral
vector with the immunoglobulin heavy chain signal
sequence under control of the CMV promoter (Figure 5).
An anti-CEA gene from the immunoZAP library is
25 then subcloned into LNC-Sig to form plasmid LNCanti-CEA.
This generates an anti-CEA variable heavy chain gene
containing a signal sequence (Figure 6). The anti-CEA
gene is first excised from immunoZAP phage DN~ as a
Bluescript SK- phagemid (see lambdaZAP protocols,
30 Stratagene, Inc. La Jolla, CA). The anti-CEA gene is
purified as an XhoI-SpeI fragment and l~gated to XhoI-
SpeI restricted LNC-S~g. LNC-Stg contains three SpeI
sites. Therefore, to generate plasmid LNCanti-CEA, the
ligation mix is transformed into neomycin-sensitive,
35 ampicillin-sensitive E. coli and neomycin-resistant,

WO93/20221 ~ 3 3 4 l i PCT/US93/02957

- 26 -
ampieillin-resistant transformants are selected for.
Plasmid LNCanti-CEA is sereened from neomycin-resistant,
ampieillin-resistant trans~ormants by using the SpeI-XhoI
anti-CEA restrietion fragment from Bluescript SK-anti-CEA
5 as a probe. The SpeI site is used because of dependenee
upon the available sites in the ImmunoZap expression
veetor. To simplify eonstruetion of LNCanti-CEA, a
unique NotI site ean be introdueed into the ImmunoZap H
expression veetor so that NotI sites ean be used instead
lO of SpeI sites.
. Construetion of LNC-immunoalobulin throuah
Plasmid pUC Star-Sig
An anti-CEA gene from the immunoZAP library is
subeloned into plasmid pUC Star-Si~ to form plasmid pUC
15 Star-anti-CEA. This generates an anti-CEA variable heavy
ehain gene eontaining a signal sequenee (Figure 12). The
anti-CEA gene is exeised from immunoZAP phage DNA as a
Blueseript SK-phagemid (~ee lambdaZAP protoeols,
Stratagene, Ine., La Jolla, CA). Blueseript SX-anti-CEA
; 20 double ~tranded DNA ~8 prQpared and restrieted with XhoI
and SpeI. The anti-CEA eontaining XhoI-SpeI f~agment is
purified by eleetroelution and ligated to Xho-SpeI
restrieted pUC Star-Sig to ereate plasmid pUC Star-
antiCEA ~Figure 12).
The antiCEA gene is transferred from pUC Star-anti
CEA to LNCX* as a HindIII-ClaI fragment to ereate plasmid
LNC-antiCEA (figure 13). The antiCEA-eontaining HindIII-
ClaI fragment is purified from pUC Star-antiCEA by
eleetroelution. Phosphatase treated, HindIII-ClaI
30 restrieted LNCX* is ligated with the purified HindIII-
ClaI antiCEA fragment to generate LNC-antiCEA ~figure
13).
Strateov for generatina targeted viruses
The starting materials for generation of targeted
35 viruses are the LNC~nv and LNC-immunoglobulin lin this

~ .

.~'`~, .
W O 93/20221 . X 1 ~ 3 4 1 1 P{~r/US93/02957
- 27 -
example, LNC-antiCEA) plasmids shown in Figure 7.
Figures 8-11 diagram the general principle for the
primary generation of targeted viruses. Hybrid
i Dunoglobulin-env proteins are generated that target
5 viruses to cells expressing carcinoembryonic antigen.
Since the location of important determinants for envelope
protein sorting (S), trimeriza~ion (T), and fusion (F) iæ
; not known with certainty, the i D unoglobulin gene is
ligated to progreæsive deletions of the env gene and
10 functional i D unoglobulin-env hybrids are selected for.
Useful Envelo~e Fraoments or Analogs
The envelope portion of the fusion protein may
consist of any portion of the envelope protein (or any
analog thereof) which is sufficient to direct efficient
15 incorporation of the envelope fusion protein into the
viral coat ~upon budding of the recombinant virus from a
producer cell line). Suc~ fragments or analogs may be
determined using the following general selection scheme
which generally involves ligation of cell surface
2Q receptor binding domains of ligand genes to progessive
deletions of viral envelope genes. The correct
combination of ligand and envelope sequences is
determined by a selection scheme for the production of
biologically active targeted virus. The selection scheme
25 not only produces targeted virus but simplifies the
construction of future targeted viruses.
Construction of hvbrid immunoalobulin-env genes in v~txo
Plasmid LNCenv contains the coding region for the
Mo-MuLV env polyprotein (Figure 8). LNCenv is first
30 linearized by HindIII restriction. A rangQ of deletions
extending into the env gene is created by colleoting
aliquots of Exonuclease III treatqd DNA over time and
removing 5'-processive ends with Sl-nuclease (Guo, I.H.,
et al., 1983, Methods Enzymol 100:60; and Sambrook, J.,
3S et al., 1989, Molecular Clonina, Cold Spring Harbor

` ~

WO93/20221 21 33 411 PCT/US93/02957

- 28 -
Laboratory Press, Cold Spring Harbor). NotI linkers (5'
AGCGGCCGCT 3 ' SEQ ID N0: 13) are ligated onto the blunt
end termini and restricted with NotI. This results in a
NotI restriction overhang at the 5'-border of every
5 deletion within the env gene. The NotI overhangs at the
other end of the molecules are removed by SalI
restriction of the reaction mixture. The reaction
mixture is then treated with phosphatase to prevent
circularization of the re~ction products.
The reaction mixture is ligated to a SalI-NotI
restriction fragment from LNC-antiCEA that contains the
~` anti-CEA variable heavy chain gene. This creates a pool
of functional retroviral vectors encoding an anti-CEA
peptide fused to a series of env deletions.
15 Generation of Dooled virus constructions
The total reaction mixture from above is
transformed into ampicillin-sensitive E. coli and
ampicillin resistance is selected for tFigure 9).
Reoombinants containing functional retroviral vectors are
20 selected for since only they contain the ampicillin
resistance gene. Plasmid DNA is prepared from~
transformants grown in liquid culture to create a pool of
retroviral vectors containing different immunoglobulin-
env fusion genes.
The DNA is tranæfected into the crip2 retroviral
packaging cell line (Danos, 0., et al., 1988 , Proc Natl
Acad Sci USA 85: 6460-6464). Alternatively, DNA is
transfected into a packaging cell line that does not
encode wild-type env protein. The transfected packaging
30 cell line synthesizes each of the different hybrid
immunoglobulin-env protein~ as well as the wild type env
protein (encoded by an env gene contained in the cell
line). The transfected packaging cell line secretes a
pool of enveloped retroviruses containing the different
35 `retroviral genomes encoding hybrid immunoglobulin-env

`` WO93/20221 ~ 1 3 3 ~ PCT/USg3/02957

- 29 -
genes. If the hybrid immunoglobulin-env protein retained
all of the neeessary determinants for efficient
incorporation into viral envelopes then the hy~rid-env
protein ean be incorporated into viral envelopes. Wild
5 type env proteins eneoded for by the packaging eell line
are also ineorporated into the viral envelopes. This
ereates a virus eontaining both wild type and hybrid env
proteins in the viral envelope. This system therefore
seleets for immunoglobulin-env hybrids that ean
10 ineorporate their gene produets into the viral envelope.
Virus pools are harvested from media filtered at
0.45~ to remove contaminating G418-resistant paekaging
eells.
Seleetion and eharaeterization of targeted virus
G418-sensitive target eells are exposed to virus
pools by standard proeedures, and G418-resistant eells
are seleeted for. The target eells ean be any non-mouse
eell line (uninfeetable by wild type No-MuLV) that
expresses eareinoembryonie antigen. Examples inelude
20 ATCC COLO 205, a human eell line isolated from the
aseites of a patient with eareinoma of the eo~on
(A.T.C.C.#CCL 222); LR-73 CEA, a ehinese hamster ovary
eell line transfeeted with a mouse eareinoembryonie
antigen gene (Benehimol, S. et al., supr~); and HCT48, a
25 human eolon adenoeareinoma eell line (Shi, 2.R., et al.,
1883, Can~er Res 43:4045-4049).
G418-resistant eells ean only have arisen from
transduetion of the neomyein resistanee gene by targeted
virus. This system therefore seleets for reeombinant
30 viruses that have hybrid immunoglobulin-~nv proteins that
have retained all the neeessary determinants for viral
targeting and fusion.
Reseue of inteorated i~munoalobulin-~nv gene
Infeetion by targeted virus results in integration
35 of the hybrid envelope gene that ereated the targeting

:

WO93/20221 PCT/US93/02~7
~1 3 ~ 411 _ 30 ~
protein. The integrated hybrid immunoglobulin-env gene
is rescued from the host DNA by polymerase chain reaction
(PCR) with the following primers:

PCR 5' Rescue primer:
5 5'-CCAGCCTCCGCGGCCCCAAGCTTCTGCA-3' (SEQ ID NO: 14)
HindIII
PCR 3' Rescue primer:
5'-GGTTC~SIaÇaAACTGCTGAGGGC-3' (SEQ ID NO: 15)
XbaI

PCR amplification with these primers generates the
immnoglobulin-env gene bordered by HindIII and XbaI
sites. The amplified DNA is restricted with HindIII and
XbaI to create sticky ends and the DNA is ligated into
HindIII-XbaI cut LNCX*. When transfected into crip2
15 packaging cells, this generates a retroviral vector
targeted to cells expressing cell surface
carcinoembryonic antigen (e.g., colon cancer cells).
The retroviral vector produced in the above
selection scheme is targeted to both CEA-expressing human
20 cells (directed by the hybrid envelope protein) and
normal mouse cells (directed by the wild type envelope
protein) when produced in crip2 packaging cells. To
create viruses that infect target cells only, the
retroviral vector will first be tested to determine if
25 incorporation of the hybrid envelope protein alone is
sufficient to direct virus fusion. This is accomplished
by transfecting DNA into a modified packaging cell line
that does not encode wild type env.
If fusion functions are found to have been
30 supplied from the wild type envelope protein, targeted
viruse~ will be created as follows. A packaging cell
line will be created that encodes an env gene containing
mutations in the receptor binding domain. When


W093~20221 PCT/US93/02g57
`~13~
- 31 -
transfected with the targeted viral vector DNA, targeted
viruses expressing both hybrid ligand-env proteins and
env proteins with mutated binding sites will be produced.
The viruses will exclusively infect target cells.
5 The taraeted viral vector is a universal vector
The viral vector that is constructed by the above
procedure is a universal targeted vector (Figure 11).
Targeting to other cells is accomplished by replacing the
XhoI-SpeI anti-CEA fragment with any XhoI-SpeI fragment
10 encoding an in-frame immunoglobulin or ligand directed
against specific cell surface proteins. For example, an
XhoI-SpeI i D unoglobulin-containing fragment from an
immunoZAP library can be fused in frame behind a signal
sequence and subcloned into LNCX* through the pUC Star-
15 Sig plasmid, as outlined above. Substituting a SalI-NotI
fragment from another LNC-immunoglobulin plasmid into the
universal vector would create another targeted virus
vector.
Other Viral Vectors
Any enveloped virus may be used as a vector for
the targeted delivery of a therapeutic gene. Particular
examples include both DNA and RNA viruses, such as
Herpesviridae, e.g., herpes simplex type 1 or 2,
Paramyxoviridae, Retroviridae, Hepadnaviridae,
25 Poxviridae, Iridoviridae, Togaviridae, Flaviviridae,
Coronaviridae, Rhabodoviridae, Filoviridae,
Orthomyxoviridae, Bunyaviridae, or Arenaviridae, or any
other, yet unclassified, enveloped virus.
An extensive selection of these viruses is
30 available, e.g., from the American Type Culture
Collection.
Taraetina Liaands
Any molecule that is capable of directing specific
interaction with a target host cell (e.g., by specific
35 recognition of and binding to a host cell surface

~ if ~
W093/20221 2133 ~i 1 i PCT/USg3/02g57

- 32 -
protein) may be used as the targeting ligand portion of
the envelope fusion protein. Preferably, such a protein
is derived from one member of a ligand:receptor pair.
The targeting ligands are not limited to proteins.
5 Carbohydrate and lipid moieties can be attached to the
envelope protein via protein fragments containing
consensus ~equences for glycosylation and lipidation.
Immunoglobulin genes can be used as ligands, as
shown in the example above. Genes for high affinity
lO immunoglobulins are screened from a lambda or bacterial
expression library by a filter binding assay with
sI] bovive serum albumin conjugated to antigen, as
previously described (Huse, et al. Sup~a ) .
Cell surface molecules such as integrins, adhesion
l5 molecules or homing receptors can be used as cell-
speaific ligands since they are involved in cell-cell
interactions via receptors on other cells. Genes
encoding these molecules can be identified by the panning
method of Seed and Aruffo (Seed. B., et al., 1987, Proc
20 Natl Acad Sci USA 84:3365-3369).
Hormones that bind to specific receptors can be
used as targeting ligands as well as viral proteins, such
as ~IV envelope protein gpl20, and modifications of
naturally occuring ligands.
25 Thera~eutic Genes
Therapeutic genes useful in the invention include
the following. l) Genes that are therapeutic to cancer
cells may include a) antisense oncogenes; b) tumor
suppressor genes, such as p53 or the retinoblastoma gene
30 product Rb, c) destructive toxin genes such as a
diphtheria toxin gene; d) cytokines æuch as tumor
necrosis factor or interferons; or e) any other
therapeutic gene. 2) Therapeutic genes targeted to
cells that are infected with HIV. Specific examples
35 include antisense DNA complementary to essential genes


` wo g3/2022l ~ 1 3 3 9 1 1 PCT/US93/02~7

- 33 -
for HIV, e.g., polymerase; destructive toxin genes, e.g.,
diphtheria toxin; and genes that will invoke
intracellular immunity, e.g., HIV enhancer sequences that
titrate and remove HIV regulatory proteins (Baltimore,
5 D., 1988, Nature 335:395-396). 3) Genes to correct
inherited deficiencies. Examples include, but are not
limited to, insulin genes delivered specifically to
pancreatic beta cells, or the cystic fibrosis
transmembrane regulator (CFTR) gene delivered to the
10 appropriate lung cells of cystic fibrosis patients. The
expression of targeted genes can be further accomplished
through the use of tissue specific enhancers that
regulate the transgene.
Therav
For any gene therapy described herein, the
appropriate recombinant virus, as described above, is
administered to a patient in a pharmaceutically-
acceptable buffer (e.g., physiological saline). The
therapeutic preparation is administered in accordance
20 with the condition to be treated. For example, to treat
an HIV-infected individual, the virus is administered by
direct injection, e.g., by intravenous, intramuscular, or
intraperitoneal injection, at a dosage that provides
suitable targeting and lysis of HIV-infected host cells.
25 Alternatively, it may be necessary to administer the
targated virus surgically to the appropriate target
tissue, or via a catheter, or a videoscope. It may be
convenient to administer the therapeutic orally, nasally,
or topically, e.g., as a liquid or spray. Again, an
30 appropriate dosage is an amount of therapeutic virus
which effects a reduction in the disease.
Targeted virus can also be administered by
implanting viral packaging cells into a patient. The
cells can be enclosed in a semi-permeable container,
35 e.g., permeable to a virus but not permeable to a

~3~4~



WO93/20221 PCT/USg3/02957


packaging cell. The implanted container may be
removable. Alternatively, the container may be hooked up
to a patient intravenously, so that virus enters the
patient through a needle or through a catheter. In this
5 way the patient receives a continuous dose of viral gene
therapy.
Other Embodiments
Other embodiments are within the following claims.
For example, replication competent viruses may be
lO used in certain cases. In other cases, where
replication-deficient viruses are necessary, it may be
efficacious to administer modified packaging cells,
rather than the targeted virus, to patients. By this
method a non-proliferating dose of recombinant virus is
15 delivered to a local area, and then the virus locates the
specific target cell. For example, tumor infiltrating
lymphocytes (TIL), which surround cancer cells, can be
modified to secrete locally high concentrations of cancer
cell-targeted virus. Treatment may be repeated as
20 necessary. Immune response against targeted viruses can
be overcome with immunosuppressive drugs.
In addition to colon cancer celLs, the ~irus of
the invention may be used to target other cancer cells,
e.g., ovarian, breast, or lung cancer cells, or cells
25 affected with hereditary diseases such a~ muscular
dystrophy, Huntington's disease, or cells with a defect
in adenosine deaminase. Herpesviridae viruses may
include Herpes simplex type 1 or type 2, Epstein-Barr
virus, or Cytomegalovirus. Sendai virus and Vaccinia
30 virus may also be adapted to this method.

~ 1 3 ~ 4 1 1
WO93/20221 . j!. PCT/US93/02~7


BBOUBNCE ~I8TING
BNERAL INFORMATION:
(i) APPLICANT: Alexander T. Young
(ii) TITLB OF IN~ENTTON: GENE THERAPY USING
TARGETED VIRAL VECTORS
(~ii) NnMBBR OF 8BQ~ENCE8: 15
(iv) CORRE8POND~NCB ADDRB88:
(A) ADDRE88~B: Fish & Richardson
(B) 8TR~T: 225 Franklin Street
~C) CITYs Boston
(D) 8TATE: Massachusetts
~B) CO~NTRY: U.S.A.
(F) 8IP: 02110-2804
~v) COMP~TBR R~ADABLB FORM:
(A) NEDI~ T~PB: 3.5" Diskette, 1.44 Nb
(B) CO~P~TDR: IBM PS/2 Model 50Z or 55SX
(C) OPDRATING 8Y8TEM: IBM P.C. DOS (Version 5.00)
(D) 8oFT~aRE: WordPerfect (Version 5.1)
~vi) C~RRENT APPLICATION DATA:
~A) APPLICATION N~NB~R:
(B) FILING DATE:
(C) C~A88IFICATION:
) PRIOR APPLIC~TION DATA:
~A) APPLI~ATIO~ N~NBER: 07/862,~95
~B) FILING DATE: April 3, 1992

(vi~) A~TO~N~Y/AG~NT INFORMATION:
~A) NAME: Paul T. Clark
(B) REGI8TRATION N~MBER: 30,162
~C) REFERBNCE/DOC~ET NUMBER:05140/002002

;~8) TELBCOMM~NICATION INFORMATION:
~A) TELBP~ON~: (617) 542-5070
~B) TE$EFAS: (617) 542-8906
(C) TELE~: 200154

t
WO 93/20221 ~13 ~ 4 ~ r/us93/o2957

- 36 -
~2) INFORMATION FOR 8BQ~ENCB IDENTIFICATION N~NBER: 1:
(i) 8BQVBNCB C~ARACTBRI8TIC8:
~A) ~BNGT~: 19
(B) TYPB: nucleic acid
~C) 8TRANDBDNB88: single
~D) TOPO~O~Y: linear
~x~) 8BQ~NCB DB8CRIPTION: SEQ ID NO: 1:

GCAGAAGGTC GACCCAACG 19
:
~2) I~FORKATION FOR 8BQV~NCB IDBNTIFICaTION NnNBBR: 2:
) 8BQV~NCB CEaRACTERI8TIC8:
:
~A) LBNGT~: 10
(B) TYPB: nucleic acid
: ~C) 8TRANDBDNB88: double
tD) TOPOLOOY: linear
Xi) 8BQV~NCB DB8CRIPTION: SEQ ID NO: 2:
.
CCAAGCTTGG 10

2) INFORNATION FOR 8EQVBNCE IDENTIFTCATION N~MBBR: 3:
~) 8BQVBNCB C~ARACTBRI8TIC8:
~A) ~EN~T~: 13
~B) TYPES nucleic acid
~C) 8TR~NDBDNB88: single
tD) TOPOLOGYs linear
~Yi) 8BQ~ ~ CB DB8CRIPTION: SEQ ID NO: 3:

GTTTTCTTTT ATC 13

~2) INFORNATION FOR 8EQVBNCJ IDENTIFICATION N~NB~a: 4:
(i) 8BQVENCB C~ARACT~RI8TIC8s
~ ~A) ~EN~T~: 15
:~ (B) ~YP~: nucleic acid
(C) 8TRAND~DNB88: single
(D) TOP0~04Y: linear



.

! --.~
9 1 1
W O ~3/20221 . PC~r/US93/02957

- 37 -
~i) 8BQUENCE DE8CRIPTION: SEQ ID NO: 4:

CAAGCATGGC TTGCC 15

(2) INFOR~ATION FOR 8EQ~ENCB IDBNTIFICATION NUNBBR: 5:
~i) 8BQ~ENCE C~RA~TERI8TIC8:
~A) LB~GTH: 22
~B) TYPE: nucleic acid
~C) 8TRaNDBDNE88: single
~D) TOPOLOGY: linear
~x~) 8BQ~BNCE DE8CRIPTION: SEQ ID NO: 5:

AGGTGCAGCT GCTCGAGTCG GG 22
(2) INFOR~ATION FOR 8BQ~BNCB IDBNTIFICATION N~MBBR: 6:
~i) 8BQ~BNCB C~ARACTBRI8TIC8:
~A) L~NG~: 22
(B) TYPE: nucleic acid
~C) 8TRANDgDNE88: single
(D) TOPOLOGY: linear
~i) 8BQ~DNCE DE8CRIPTION: SEQ ID NO: ~:

AGGTGCAACT GCTCGAGTCT GG 22

(2) INFORMATION FOR 8EQ~NCE IDENTIFICATION N~NBER: 7:
(i) 8~QUENCE C~ARACTERI8TIC~:
~A) L~NGT~: 22
(B) TYPE: nucleic acid
~C) 8TRANDEDNE88: single
~D) TOPOLO~Y: linear
~xi) BEQ~BNCB DE~CRIPTION: SEQ ID NO: 7:

AGGTGCAGCT GCTCGAGTCT GG 22

W O 93/20221 . ~ ~3~ PC~r/US93/02957

- 38 -
~2) INFO~NATION FOR 8~Q~NC~ IDENTIFICATION NUMBER: 8:
(i) 8EQ~ENCE C~ARACTBRI8TIC8:
~A) LENGTa: 22
(B) TYP~s nucleic acid
~C) 8TRANDEDN~88: single
(D) TOPOLOaYs linear
(~i) 8BQ~BNCB DE8CRI~TION: SEQ ID NO: 8:

~: AGGTGCAACT GCTCGAGTCT GG 22

~2) INFOR~ATION FOR 8BQ~ENCB IDENTIFICATION N~MBER: 9:
(1) 8EQ~ENCB C~ARACT8RI8TIC8:
~A) LENGT8: 22
~B) TYPB: nucleic acid
~C) 8TRANDEDNB88s single
~D) TOPO~O~Y: linear
: (xi) 8BQ~BNCB DE8CRIPTION: SEQ ID NO: 9:

AGGTCCAGCT GCTCGAGTCT GG 22
: ~ ~2) INFORMATION FOR 8EQ~BNCE IDENTIFICATION NWMBBR: 10:
~:~ (i) 8BQ~BNCB C~ARACTERI8TIC8:
~A) ~ENGT~: 39
~B) TYPB: nucleic acid
~C) 8TaANDBDNE88s single
~D) TOPOLOCY: linear
~i) 8~QVENCE DE8CRIPTION: SEQ ID NO: 10:

CTATTAACTA GTGACGGTTA CCGTGGTCCC TTGGCCCCA 39

t2) INFORMATION FOR 8EQVgNCE IDENTIFICATION N~MB~R:
11:
~i) 8~QVENC~ C~RACTERI8TIC8:
~A) ~ENGT~: 45
(B) TYP~: nucleic acid
(C) 8~RAND~DNE88: double
~: (D) TOPOLOGY: linear
:

I` i~13~4~J 1
WO 93/20221 , . PCI~/US93/02957

- 39 -
~xi) 8gQ~NCE Dg8CRIPTION: SEQ ID NO: 11:

AGCTTCTGCA GGCTCGAGTG ATCAACTAGT GCGGCCGCAT CGATT 45

(2) INFORNATION FOR 88Q~BNCg ID8NTIFICATION NVMBER: 12:
(i) 8BQ~BNCB C~ARACTBRI8TIC8:
~A) LB~T~: 4s
~B) TYP8: nucleic acid
tC) 8TRANDEDNE88: double
tD) TOPOLOGY: linear
tx~) 8gQ~NCB DB8CRIPTION: SEQ ID NO: 12:

AGACGTCCGA GCTCACTAGT TGATCACGCC GGCGTAGCTA AGATC 45

(2) INFORKaTION FO~ 8BQ~E~CE IDENTIFICATION N~NBER: 13:
i) 8BQ~NC~ C D ACTBRI8TIC8:
~A) LBN~T~: 10
(B) TYPBs nucleic acid
tC) 8T~A~D8DN888: double
(D) TOPOLO~Ys linear
~: (x~) 8BQ~NCB DB8CRIPTION: SEQ ID NO: 13:

~GCGGCCGCT 10

t2) INFOR~TION FOR 8BQ~NCB IDBNTIFICATION N~MB~R: 14:
(~) 8BQ~BNCB C~ARACTERI8TIC8:
~A) LENGT~: 28
(B) TYPE: nucleic acid
~C) 8TRANDEDNE88: single
(D) TOPoLoaY: linear
~i) 8EQ~NCE D~8CRIPTION: SEQ ID NO: 14:

CCAGCCTCCG CGGCCCCAAG CTTCTGCA 28



.

WO93/20221 . '1 ~3 ~ PCT/US93/02957

- 40 -
~2) INFOR~ATION FOR 8~QU~NCE ID~NTIFICATION NUMBDR: l5:
~) 8EQ~ENC~ C~ARACT~RI8TIC8:
~A) LEN~T~: 24
~B) TYPF: nucleic acid
~) 8TRANDFDNB88: single
~D) TOPO~OGY: linear
~i) 8~Q~ENCE DE8CRIPTION: SEQ ID NO: 15:

GGTTCTCTA GAAACTGCTG AGGGC 24

Representative Drawing

Sorry, the representative drawing for patent document number 2133411 was not found.

Administrative Status

For a clearer understanding of the status of the application/patent presented on this page, the site Disclaimer , as well as the definitions for Patent , Administrative Status , Maintenance Fee  and Payment History  should be consulted.

Administrative Status

Title Date
Forecasted Issue Date Unavailable
(86) PCT Filing Date 1993-03-31
(87) PCT Publication Date 1993-10-14
(85) National Entry 1994-09-30
Examination Requested 2000-02-04
Dead Application 2003-03-31

Abandonment History

Abandonment Date Reason Reinstatement Date
1998-03-31 FAILURE TO PAY APPLICATION MAINTENANCE FEE 1998-04-29
2002-04-02 FAILURE TO PAY APPLICATION MAINTENANCE FEE

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Application Fee $0.00 1994-09-30
Maintenance Fee - Application - New Act 2 1995-03-31 $50.00 1995-02-15
Reinstatement: Failure to Pay Application Maintenance Fees $200.00 1996-05-27
Maintenance Fee - Application - New Act 3 1996-04-01 $50.00 1996-05-27
Maintenance Fee - Application - New Act 4 1997-04-01 $50.00 1997-03-24
Reinstatement: Failure to Pay Application Maintenance Fees $200.00 1998-04-29
Maintenance Fee - Application - New Act 5 1998-03-31 $150.00 1998-04-29
Maintenance Fee - Application - New Act 6 1999-03-31 $150.00 1999-03-22
Request for Examination $200.00 2000-02-04
Maintenance Fee - Application - New Act 7 2000-03-31 $150.00 2000-03-21
Maintenance Fee - Application - New Act 8 2001-04-02 $150.00 2001-03-29
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
YOUNG, ALEXANDER T.
Past Owners on Record
None
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Cover Page 1995-08-26 1 26
Description 1995-08-26 40 2,291
Abstract 1995-08-26 1 41
Claims 1995-08-26 5 193
Drawings 1995-08-26 14 367
Fees 2001-03-29 1 40
Assignment 1994-09-30 4 177
PCT 1994-09-30 11 388
Prosecution-Amendment 2000-02-04 1 45
Correspondence 2000-02-04 1 20
Prosecution-Amendment 2000-03-07 2 107
Fees 1996-05-08 4 183
Fees 1997-03-24 1 54
Fees 1996-05-27 1 71
Correspondence 1996-05-27 1 23
Fees 1995-02-15 1 39