U.S. patent application number 10/244715 was filed with the patent office on 2003-05-08 for use of ribozymes for functionating genes.
Invention is credited to Keck, James G., Wong, Justin G.P..
Application Number | 20030087288 10/244715 |
Document ID | / |
Family ID | 21818282 |
Filed Date | 2003-05-08 |
United States Patent
Application |
20030087288 |
Kind Code |
A1 |
Keck, James G. ; et
al. |
May 8, 2003 |
Use of ribozymes for functionating genes
Abstract
Methods and compositions for identifying a gene or genes
associated with the generation of a specific cellular phenotype or
a specific cellular response using combinatorial libraries of
catalytic RNA directed against RNA sequences encoding structural or
functional polypeptide motifs. The invention is exemplified by use
of a combinatorial ribozyme library to target sequence in mRNAs
encoding zinc finger, protein kinase and integrin motifs.
Inventors: |
Keck, James G.; (Redwood
City, CA) ; Wong, Justin G.P.; (Oakland, CA) |
Correspondence
Address: |
HELLER EHRMAN WHITE & MCAULIFFE LLP
4350 LA JOLLA VILLAGE DRIVE
7TH FLOOR
SAN DIEGO
CA
92122-1246
US
|
Family ID: |
21818282 |
Appl. No.: |
10/244715 |
Filed: |
September 16, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10244715 |
Sep 16, 2002 |
|
|
|
09601970 |
Aug 9, 2000 |
|
|
|
09601970 |
Aug 9, 2000 |
|
|
|
PCT/US99/03166 |
Feb 12, 1999 |
|
|
|
10244715 |
Sep 16, 2002 |
|
|
|
09023992 |
Feb 13, 1998 |
|
|
|
Current U.S.
Class: |
506/10 ;
435/6.14; 435/6.16; 435/7.1; 506/14; 506/16; 506/17; 506/18;
506/26 |
Current CPC
Class: |
C12N 15/1034 20130101;
C12N 15/113 20130101; C12N 2799/021 20130101; C12N 2310/121
20130101; C12N 2799/027 20130101; C12N 2310/111 20130101 |
Class at
Publication: |
435/6 ;
435/7.1 |
International
Class: |
C12Q 001/68; G01N
033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 15, 1999 |
IN |
188/MAS/99 |
Claims
What is claimed:
1. A method for identifying one or more members of a combinatorial
ribozyme library that alters a phenotype of a host cell, said
method comprising the steps of: growing a cell culture comprising
host cells, wherein said host cells comprise a transcription
product of a mammalian target nucleic acid encoding a motif of
interest, wherein the nucleotide sequence of said mammalian target
nucleic acid is unknown; contacting said cell culture with a
plurality of members of a combinatorial ribozyme library, which
bind to said transcription product, whereby expression of said
transcription product is disrupted, wherein: each member of library
comprises a catalytic domain that cleaves a sequence within the
transcription product of the target nucleic acid that encodes a
motif of interest or variant thereof; and the library comprises a
plurality of such members; identifying host cells that exhibit one
or more phenotypic changes, whereby said host cells exhibiting one
or more phenotypic changes are isolated, whereby said one or more
members of said combinatorial library are identified.
2. The method of claim 1, further comprising the step of; utilizing
the nucleotide sequence of said one or more members of said library
that disrupt expression of said transcription product as a probe to
identify nucleotide sequences of said transcription product and
said mammalian target nucleic acid, whereby said nucleotide
sequences of said transcription product and said mammalian target
nucleic acid are obtained.
3. The method of claim 1, wherein said host cell culture comprises
a plurality of mammalian cells, bacterial cells, invertebrate cells
or plant cells.
4. The method of claim 1, wherein said motif is a zinc finger
motif, a receptor protein kinase motif, or an integrin motif.
5. The method of claim 1, wherein said transcription product is
mRNA.
6. A double stranded DNA molecule, comprising: a sense strand and
an antisense strand, wherein said sense strand codes for a
catalytic domain, which when expressed as RNA, disrupts expression
of an mRNA transcribed from a target nucleic acid encoding a motif
of interest and binding regions flanking said catalytic domain for
binding said RNA to said mRNA, and wherein a means for determining
directionality of expression is included in said double stranded
DNA.
7. A vector comprising a double-stranded DNA of claim 6.
8. The vector of claim 7, wherein said double-stranded DNA further
comprises regulatory elements for expression.
9. The double stranded DNA of claim 6, wherein said means for
determining directionality of expression comprises a different non
blunt-ended restriction enzyme site at each end of said double
stranded DNA.
10. The double stranded DNA of claim 6, wherein said RNA is a
ribozyme.
11. A retrovirus expression vector comprising: a retrovirus plasmid
vector comprising a double stranded DNA of claim 6.
12. The retrovirus expression vector of claim 11, wherein said
vector comprises supercoiled DNA.
13. A retrovirus packaging cell line comprising: a retrovirus
expression vector of claim 11.
14. An adeno-associated virus expression vector comprising: an
adeno-associated virus plasmid vector comprising a double stranded
DNA of claim 6.
15. An adeno-associated virus packaging cell line comprising: an
adeno-associated virus expression vector of claim 14 and an
adeno-associated virus helper plasmid.
16. A plasmid expression vector comprising: a double stranded DNA
of claim 6.
17. A combinatorial library comprising: a plurality of
double-stranded DNA molecules of claim 6, wherein said binding
regions are degenerate.
18. The combinatorial library of claim 17, wherein said target
nucleic acid encodes a protein.
19. The combinatorial library of claim 18, wherein said protein is
an enzyme.
20. The combinatorial library of claim 28, wherein said enzyme is a
protein kinase or a protease.
21. The combinatorial library of claim 18, wherein said protein
contains a zinc-finger domain.
22. The combinatorial library of claim 18, wherein said protein
contains an integrin domain.
23. The combinatorial library of claim 18 wherein said protein is a
signaling molecule or a structural protein.
24. The double stranded DNA of claim 6, wherein said motif is a
zinc finger motif, a protein kinase motif or an integrin motif.
25. A retrovirus particle, comprising: a genome encoding an RNA
comprising a catalytic domain that cleaves mRNA transcribed from a
target nucleic acid encoding a motif of interest, and binding
sequences flanking said catalytic domain for binding said RNA to
said mRNA.
26. A mammalian cell, comprising: one or more double stranded
DNA(s) comprising a sense strand and an antisense strand, wherein
said sense strand codes for a catalytic domain, which when
expressed as RNA, cleaves a mRNA sequence transcribed from a target
nucleic acid encoding a motif of interest, and binding regions
flanking said catalytic domain for binding said RNA to said mRNA,
and wherein a means for determining directionality of expression is
included in said one or more double stranded DNA(s).
27. A adeno-associated virus, comprising: a genome encoding of an
RNA comprising a catalytic domain for cleavage of an mRNA
transcribed from a target nucleic acid comprising a sequence
encoding a structural motif of interest, and binding regions
flanking catalytic domain for binding said RNA to said mRNA.
28. A ribozyme, comprising: a catalytic domain and binding domains
complementary to a nucleotide sequence encoding a motif of
interest, that disrupts expression of said nucleotide sequence.
29. The ribozyme of claim 28, wherein said nucleotide sequence is
RNA.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 09/601,970, filed Aug. 9, 2000. U.S. application Ser. No.
09/601,970, is the national stage of International PCT application
No. PCT/US99/03166, filed Feb. 12, 1999. This application is a
continuation-in-part of U.S. application Ser. No. 09/023,992, filed
Feb. 13, 1998. The subject matter of these applications is
incorporated by reference in its entirety.
INTRODUCTION
[0002] 1. Technical Field
[0003] The present invention is related to methods and compositions
for identifying a gene or genes associated with the generation of a
specific cellular phenotype or a specific cellular response using
combinatorial libraries of catalytic RNA directed against RNA
sequences encoding structural or functional polypeptide motifs. The
invention is exemplified by use of a combinatorial ribozyme library
to target sequence in mRNAs encoding zinc finger, protein kinase
and integrin motifs.
[0004] 2. Background
[0005] Properly functioning cells are necessary for any organism,
including humans, to thrive; improperly functioning cells may
contribute to the development of pathogenic or disease states in a
given individual, including generation of cancers, autoimmune
diseases, innate immunodeficiencies, neurologic diseases, and
inborn errors of metabolism. In addition, even properly functioning
cells may contribute to pathogenic states, including susceptibility
to infectious agents, atopic/allergic pathogeneses, and pathogenic
states associated with allograft transplantation. In both of the
above cases, inappropriate expression, regulation, or function of a
specific gene product or gene products within a cell may lead to
the improper behavior of that cell within the context of its normal
function in an organism. Often, the activity of a single gene
product, such as a protein or polypeptide, will affect the
expression, regulation, or function of other gene products within
the same cell or within neighboring cells. Aberrant expression,
regulation, or function of these aggregated gene products may then
result in the development of specific disease phenotypes or
syndromes.
[0006] Approaches that have been used to identify genes which are
potentially involved in a disease development process include
identification of genes which are mutated in certain diseases and
differential display of actively expressed transcripts in normal
versus pathologic cells. These approaches have given rise to a
rapid increase in the number of DNA sequences associated with
various pathologic states. These sequences include not only full
length genes, but also cDNA sequences comprised of partial gene
sequences or ESTs. Although sequences identified by these processes
are associated with a pathologic state, it is difficult to
ascertain a priori whether a given gene is directly involved in the
disease development process, or whether its expression occurs in a
secondary fashion after the pathogenic process has already
begun.
[0007] Involvement of particular genes as causative agents in the
disease development process can be confirmed by a number of
methods. Confirmation of the role of particular genes in the
disease development process using partial cDNA sequences is more
difficult to assess, however, because many of the methods used
require knowledge of the full gene sequence. Thus, while the number
of potentially novel genes has expanded exponentially,
identification of the functions ascribed to most of these genes and
gene sequences, as well as their prospective roles in disease
development has lagged behind.
[0008] One way to establish the causative effect of a gene or gene
sequence in the development of a specific cellular phenotype or
response is to interfere with the expression or function of that
gene or gene product, and then to determine the resulting effect on
that cellular phenotype or response. Methods utilized to interfere
with gene expression in vivo involve gene targeting by homologous
recombination in embryonic stem cells, re-implantation of the stem
cells, gestation of the embryos, and isolation of animals bearing
diallellic deletions in the gene of interest, so called "transgenic
technology". The development of transgenic technology has been an
important advance in the tools available for studying the function
of genes at the organismal level. Because this procedure can take
up to a year to complete, however, it is not an efficient process
for the high-throughput evaluation of genes or gene products as
causative agents and as potential therapeutic targets. Methods
utilized to interfere with gene expression in vitro include gene
deletion or inactivation by homologous recombination or triplex
technology, RNA transcript inactivation or cleavage by antisense or
ribozyme technology, and protein inactivation or down-regulation by
antipeptide antibody fragments or expression of randomized
peptides. A limitation to utilizing systems expressing randomized
peptides, antisense RNA molecules, or anti-peptide antibodies to
identify gene functions and/or signaling pathways in cells is that
these compounds do not act catalytically as is the case for
ribozymes and therefore, relatively high intracellular
concentrations may be necessary to affect a cellular function or
phenotype.
[0009] Ribozymes are RNA molecules that act as enzymes and can be
engineered to cleave other RNA molecules. Thus, ribozymes perform
functions in the cell that are very different from ordinary RNA, in
that, after binding selectively to their specific mRNA target, they
act catalytically to cut, or cleave, target RNA molecules at
specific sites. If an mRNA target in a cell is destroyed, the
particular protein for which mRNA molecule carries information is
not produced. The ribozyme itself is not consumed in this process,
and can act catalytically to cleave multiple copies of mRNA target
molecules. One way to use ribozymes to identify the function of
novel gene sequences is to introduce a pool of ribozymes with
degenerate target recognition sites into cells in order to reduce
or eliminate the expression of a gene or gene product involved in
the generation of a specific cellular phenotype or response. In
this strategy, ribozymes bearing the appropriate recognition
sequences eliminate or reduce expression of the target gene, while
ribozymes not bearing the appropriate recognition sequences do not.
Loss of a specific cellular phenotype or response associated with
elimination or reduction in expression of a target gene indicates
involvement of that particular gene in the development of that
particular phenotype or response.
[0010] Of the estimated 100,000 expressed genes in a mammalian
cell, approximately one-third are likely to be necessary for normal
cell respiration, metabolism, or viability. A totally degenerate
ribozyme library would by necessity include ribozymes directed
against these "housekeeping genes" as well as against genes
involved in disease processes. Cleavage of housekeeping RNAs
results in compromised cellular viability, so no information can be
gained from a great number of the ribozyme sequences in such a
library. This problem reduces the efficiency of using totally
degenerate ribozyme libraries to identify and assign a function to
novel genes or gene sequences with respect to a disease development
process. Another major limitation to this system is the need to
synthesize and express a completely randomized library of nucleic
acids and to screen the library for functional activity. The
minimal targeting or recognition sequence of a ribozyme is
generally 12 nucleotides and a totally random library would contain
412 or approximately 16 million ribozymes. Due to the large number
of permutations of the ribozyme binding sequences, a specific
targeting approach is essential. It is therefore of interest to
develop a high throughput ribozyme based screening system that
limits the potential target sequences for evaluation to those which
have an increased probability of being associated with a molecular
pathway that is related to a disease or phenotype.
[0011] Relevant Literature
[0012] An RNA molecule not naturally occurring in nature having
enzymatic activity independent of any protein is disclosed in U.S.
Pat. No. 4,987,071. General rules for the design of hammerhead
ribozymes that cleave target RNA in trans are described in Haseloff
and Gerlach, (1988) Nature 334:585-591. Miniribozymes are disclosed
in Uhlenbeck, (1987) Nature 328:596-603. Methods for optimizing
cleavage of a target RNA by a ribozyme are described in U.S. Pat.
No. 5,496,698. Reporter gene suppression by engineered hammerhead
ribozymes in mammalian cells is described in Cameron and Jennings
(1989) Proc. Natl. Acad. Sci. (USA) 86:9139-9143. Ribozyme
expression from a retroviral vector is described in Sullenger and
Cech, (1993) Science 262:1566-1569. The expression of hammerhead
ribozymes operatively linked to a T7 promoter is described in
Chowrira et al, (1994) J. Biol. Chem. 269:25856-25864.
Co-localizing ribozymes with substrate RNAs to increase their
efficacy as gene inhibitors is described in Sullenger, (1995) Appl.
Biochem. Biotechnol. 54:57-61. Screening of retroviral cDNA
expression libraries is described in Kitamura, et al., (1995) Proc.
Nat. Acad. Sci. (USA) 92-9146. Selection of efficient cleavage
sites in target RNAs by using a ribozyme expression library is
described in Lieber and Strauss, (1996) Mol. Cell. Biol.
15:540-551. Approaches for the identification and cloning of
differentially expressed genes is discussed in Soares, (1997) Curr.
Opin. Biotechnol. 8:542-546. The development of high-throughput
screen is discussed in Jayawickreme and Kost, (1997) Curr. Opin.
Biotechnol. 8:629-634. The high throughput screen for rarely
transcribed differentially expressed genes is described in von
Stein et al., (1997) Nucleic Acids Res. 25:2598-2602.
High-throughput genotyping is disclosed in Hall, et al., (1996)
Genome Res 6:781-790. Methods for screening transdominant
intracellular effector peptides and RNA molecules are disclosed in
WO97/27212 and WO97/27213.
SUMMARY OF THE INVENTION
[0013] Methods and compositions for their use therein, are provided
for determining and validating a link between a target nucleic acid
which includes a nucleotide sequence that encodes a motif of
interest and a disease and/or phenotype using a combinatorial
ribozyme library. Ribo-nucleotide members of the ribozyme library
include a binding region which is complementary to a transcription
product of the target nucleic acid and a catalytic domain which
cleaves a sequence within a transcription product of the target
nucleic acid coding for the motif of interest so that expression of
the transcription product is disrupted. The method includes the
steps of designing a combinatorial ribozyme library by analyzing a
consensus nucleotide sequence encoding a protein motif and
synthesizing members of a library of sense strands of DNA which,
when expressed as RNA constitute the members of a ribozyme library;
annealing the sense strands to antisense strands to form double
stranded DNAs, introducing the double stranded DNAs, which
optionally include a means for determining directionality of
expression, into expression vectors; contacting a host cell culture
containing one or more host cells with the expression vector(s)
under conditions such that the expression vectors transfect or
infect the host cells; growing the host cells to express the
ribozyme(s); analyzing the phenotype of, or a suitable detectable
marker in, the resultant transfected or infected host cells to
identify any altered host cell by virtue of an alteration in
phenotype or marker as compared to unmodified host cells; isolating
altered host cells; and correlating the phenotype of altered host
cells with the identity of the target nucleic acid encoding the
motif of interest by isolating DNA from the isolated altered host
cell and determining the specific ribozyme sequence contained in
the isolated DNA which is complementary to sequences in the target
nucleic acid so as to assign a function to the product coded for by
the target nucleic acid. The ribozyme libraries and subject methods
can be used, for example, for functionating a gene encoding a
protein that contains a motif of interest, such as a gene involved
in apoptosis, drug susceptibility, cell cycle regulation, cell
differentiation or transformation of a host cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows the general structure of the members of a
combinatorial ribozyme library annealed to an mRNA encoding the
minimal recognition sequence of the reverse translated zinc finger
motif (SEQ ID NO: 43), C-X-X-C (X=any amino acid). Upper strand
(SEQ ID NO:1) is the targeted mRNA with the ribozyme cleavage site
indicated. The lower stand (SEQ ID NO:2) is a hammerhead ribozyme
annealed to the mRNA target. (N=any nucleotide).
[0015] FIG. 2 shows the nucleotide sequence of oligonucleotides
encoding an anti-EGFP hammerhead ribozyme (SEQ ID NOS:3-22).
[0016] FIG. 3 demonstrates the isolation of cells expressing a
selectable marker associated with a ribozyme-expressing construct
from Jurkat T-cell cultures transduced with a library of ribozymes.
The selectable marker is the cell surface molecule Lyt-2 (CD8a).
Cells expressing the Lyt-2 marker are isolated from the rest of the
population using a fluorescence activated cell sorter. The X axis
depicts marker expression. The Y axis depicts cell number. The
histogram in FIG. 3A shows the profile of marker expression in
transduced cultures. The histogram in FIG. 3B shows the same
histogram with an expanded Y axis to reveal the marker positive
population shifting rightward in the histogram. Marker positive
cells isolated by flow cytometric cell sorting were grown in
culture, and marker expression was re-analyzed in the enriched
cultures. The histogram in FIG. 3C shows results from this
re-analysis. All cells in the enriched cultures express the marker,
demonstrating the ability to isolate a stable population of cells
expressing a library of pooled ribozymes using this method.
[0017] FIG. 4 demonstrates the effect of expressing a library of
ribozymes on the induction of a cellular protein by cells in a
culture. Loss of the ability to induce the protein exemplifies the
loss of a cellular response in ribozyme-expressing cells. The X
axis depicts expression of the induced protein. The Y axis depicts
cell number. The histogram in FIG. 4A shows the profile of induced
protein expression in normal cultures (stippled lines) or in
cultures expressing a library of pooled ribozymes (solid lines).
The histogram in FIG. 4B shows the same histogram with an expanded
y-axis to reveal the leftward shifting population of cells,
corresponding to those cells which have lost the ability to induce
the protein. Cells from the leftward part of the histogram in FIGS.
3A and B were isolated by flow cytometric cell sorting, grown in
culture, and induction of the cellular protein was re-analyzed. The
histogram in FIG. 4C demonstrates that the subpopulation of cells
which have lost the responsive phenotype (represented by the
left-hand peak of the histogram) can be enriched from cultures
expressing several different ribozyme species represented in the
original pooled library.
BRIEF DESCRIPTION OF PREFERRED EMBODIMENTS
[0018] In the present invention, a combinatorial ribozyme library
designed for a target nucleic acid, DNA or RNA, that contains a
nucleotide sequence encoding a motif of interest is developed and
used as a means of assigning a function to the target nucleic acid.
The term "ribozyme" is intended to mean a synthetic RNA molecule
that acts as an enzyme and has been engineered to cleave other RNA
molecules; after binding selectively to a specific RNA target
molecule, it acts catalytically to cut, or cleave, a specific RNA
target molecule in a region encoding a motif such as a zinc finger,
a protein kinase or an integrin. Ribonucleotide members of the
ribozyme library include a binding region which is complementary to
a transcription product of the target nucleic acids and a catalytic
domain which cleaves a sequence within a transcription product of
the target nucleic acid coding for the motif of interest so that
expression of the transcription product is disrupted. The binding
region generally flanks the catalytic domain. The ribozyme library
is introduced into a viral vector such as a retrovirus vector or a
plasmid vector which is then used to infect or transfect a host
cell culture that is grown to express the ribozyme library;
depending upon the system used, the vector can be incorporated into
the host cell genome or can be episomal. Optionally, the DNA of the
vector is supercoiled. The host cell culture includes at least one
host cell and can contain a plurality of host cells. The host cell
generally is a mammalian cell but can be a lower or higher plant
cell, an invertebrate cell or a bacterial cell. The expression of
the ribozyme in the host cell alters the phenotype of the host cell
so that a function for the product encoded by the target nucleic
acid can be assigned based upon the change in phenotype. The term
"function" is intended to mean a detectable or measurable event.
The target nucleic acid encodes an expression product that is
directly or indirectly involved in a measurable function or
phenotype in a host cell containing the target nucleic acid.
Generally the expression product is a protein, including signaling
molecules and structural proteins. The term "motif" is intended to
mean a conserved or partially conserved sequence shared by a
functionally or structurally related class or family of proteins.
The term "phenotype" is intended to mean a characteristic of a
specific cell or cell population and includes physical functions
such as membrane permeability, physiological functions which
include those affected directly or indirectly by regulatory
effectors, and biochemical and biological characteristics and
functions such as protein synthesis and enzyme activity. The host
cell exhibiting an altered phenotype is identified using and
isolated using any of a variety of standard techniques. DNA coding
for the ribozyme is identified in the DNA isolated from the host
cell, conveniently by PCR amplification of the mRNA or genomic DNA
coding for the ribozyme using a primer pair derived from vector
sequences flanking the ribozyme insert. The PCR product is then
sequenced to obtain the sequence of the ribozyme-coding sequence,
which not only identifies the biologically active ribozyme, but
also the identity of the target nucleic acid.
[0019] There are several advantages to the subject invention. By
targeting the combinatorial ribozyme library to conserved or
partially conserved motifs associated with known functions or
properties of proteins or polypeptides containing such motifs, the
number of ribozymes that need to be constructed and analyzed is
significantly reduced (less than about 100,000) in comparison to a
random library (over 16 million). The ability to eliminate the step
of amplifying plasmid DNA in bacteria such as E. coli is a major
cost saving advantage as well as a time saving advantage over
existing technologies: removal of the E. coli amplification can
subtract several labor intensive days from the entire process.
Furthermore, the subject process lends itself to automation when
implemented in a matrix format or a 96-well or similar multi-well
format. The simultaneous construction, delivery and expression of
multiple members of a combinatorial ribozyme library and their
analysis offers the advantage that a large number of ribozymes can
be expressed conveniently in host cell cultures, thereby enabling
the identification of genes and determining the function of genes
by a manageable high throughput screening process in a relatively
short period of time. Furthermore, the combinatorial ribozyme
library can be constructed with synthetic oligonucleotide DNA which
offers the additional advantage that directionality is conveniently
achieved by incorporating unique restriction enzyme sites at both
ends of each of the oligonucleotides used to prepare the
double-stranded DNA coding for these molecules so that
double-stranded DNA is ligated to the delivery vector in the
correct orientation for expression. This overcomes the problem that
if the same restriction enzyme site, blunt ends or restriction
enzyme sites comprising compatible cohesive ends are used for the
ligation, theoretically about 50% of all the constructs would be
ligated in the incorrect orientation. Other advantages of the
subject invention include the capability to regulate the magnitude
and timing of nucleic acid expression and high throughput delivery.
Operatively linking the oligonucleotide DNAs encoding the
combinatorial ribozyme library to a regulatable promoter provides
temporal and/or cell type specific control throughout the screening
assay. Additionally, the magnitude of ribozyme expression can be
modulated using promoters that differ in their transcriptional
activity.
[0020] Ribozyme technology in particular offers several advantages
over other methods used to determine which genes are relevant to a
disease because as used in the subject invention they are selective
for a specific target motif sequence and act catalytically, rather
than in a stoichimetric manner. Thus, a single ribozyme molecule
can cleave and inactivate up to 100 RNA transcripts, while a single
antisense or antipeptide molecule will only inactivate one RNA
transcript or one polypeptide. These properties can be used to
identify the role of a target genetic sequence and to characterize
its cellular function and the function of its encoded product. In
the disclosed invention, it is not necessary to develop
conformational models of the target nucleic acids to identify
regions which are particularly accessible. Such models typically
are developed using computer-assisted predictions of possible
thermodynamically stable secondary structures. The need for such
computer generated models is avoided by creating a combinatorial
ribozyme library targeted to nucleic acids encoding a motif of
interest. Additionally, sustained expression of ribozyme activity
can be achieved by utilizing plasmid or viral based expression
constructs driven by cellular promoters in order to constitutively
express high levels of ribozymes directed against the target of
interest, ensuring sufficient levels of cellular genes are
inactivated to cause a detectable change in cellular phenotype or
response.
[0021] Another advantage to using ribozyme molecules for
inactivation of cellular RNA transcripts is that recognition of an
mRNA target by the ribozyme molecule requires the complementary
base-pairing of only 12-14 nucleotides. Knowledge of the entire
sequence of the gene of interest is therefore not necessary. This
characteristic, together with the aforementioned ability of
ribozymes to function catalytically makes them useful for
identifying the roles of genes where only partial sequences are
known, as well as the roles of genes where the full length sequence
is known.
[0022] By constructing combinatorial ribozyme libraries bearing
recognition sites derived from DNA or RNA sequences encoding known
protein functional motifs, the likelihood that a ribozyme in the
library will cleave a transcript involved in a "functional" gene is
greatly increased. An additional advantage to this strategy is that
more than one combinatorial library can be introduced into host
cells simultaneously, allowing isolation of genes containing,
combinations of specific motifs, which contributes to specificity
of the ribozyme for a particular gene. For example, one can isolate
with equal ease "genes which are transmembrane protein receptors
with intracellular tyrosine kinase domains and SH2 regions" as well
as "all genes with kinase function."
[0023] The combinatorial ribozyme library is designed by analyzing
a consensus nucleotide sequence coding for a protein motif of
interest. Motifs of interest are identified by use of scientific
literature; public and/or private databases; and other sources
(e.g., Prosite: http://expasy/hcuge.ch/) that contain information
regarding the relatedness of various proteins based on amino acid
sequence homology. Proteins with one or more shared function or
class tend to contain similar amino acid patterns or motifs that
are common for each class of protein. For example, receptor
tyrosine kinases, enzymes involved in the transfer of phosphate to
tyrosine residues on protein substrates, often contain the amino
acid sequence: G-X-H-X-N-[LIVM]-V-N-L-L-G-A-C-T (SEQ ID NO:23)
wherein X=any amino acid, and [ ]=containing only one of the amino
acids listed within the brackets. Examples of tyrosine kinases that
contain this sequence are platelet-derived growth factor,
macrophage colony stimulating factor receptor (fms oncogene), stem
cell factor receptor (kit oncogene), and vascular endothelial
growth factor (VEGF) receptors Flt-1 and Flk-1/KDR. These molecules
have been demonstrated to participate in various signal
transduction pathways.
[0024] The subject invention is designed to identify molecules,
previously known or unknown, to have comparable roles in the
function of a host cell(s) and to be specifically associated with
disease states or phenotypes. Other examples of conserved motifs
that are contained in functionally related classes of proteins that
are critical for cell function are proteases. For instance,
caspase-1, known as interleukin-1 beta converting enzyme (ICE),
represents a family of proteases (caspase-1 to 12) involved in
apoptosis which has the consensus motifs
K-P-K-[LIVMF](4)-Q-A-C-[RQG]-G (SEQ ID NO:24) and
H-X(2,4)-[SC]-X(4)-[LIV- MF](2)-[ST]-H-G (SEQ ID NO:25). For
abbreviations, see supra. Caenorhabditis elegans, ced-3, and
Drosophila ICE also contain these motifs.
[0025] Other motifs are shared by proteins that have a common
structural relationship. For example, the zinc finger motif has
been found in a variety of DNA-binding proteins. One zinc finger is
known as the C3HC4 domain and has the consensus sequence:
C-X-H-X-[LIVMFY]-C-X(2)-C-[LIVMYA] (SEQ ID NO:26). This motif is
found in a diverse range of proteins including the BRCA1 protein
that is associated with breast cancer, protein RAG-1 that is
involved in rearrangement of immunoglobulin and T-cell receptor
genes and in RO/SS-A which is associated with lupus and Sjogren's
syndrome. Another example is a portion of the integrin family that
has the conserved sequence: G-X-[GNQ]-X(1,3)-G-X-C-X-C-X(2)-C-X-C
(SEQ ID NO: 27). The integrins are involved in cell to cell and
cell to matrix adhesion: cellular functions that may be important
in metastasis and tumor invasion.
[0026] Motifs found in protein kinases, integrins, caspases and
zinc-finger domains have been described. The combinatorial ribozyme
library, however, can be designed to target the mRNA encoding any
protein for which a conserved sequence can be identified. These
include enzymes such as proteases, structural proteins and
signaling molecules.
[0027] Different regions within the same motif can be targeted. In
addition, if a family or class of proteins contains more than one
motif, multiple motifs also can be targeted. The targeted motifs
are not limited to those found in proteins with known mammalian
regulatory functions but also can be motifs that have only been
identified in other organisms such as yeast, Drosophila,
Caenorhabditis elegans. Therefore, human genes critical to disease
processes or phenotypes that encode proteins containing motifs
similar to those in genes in lower eukaryotes can be
identified.
[0028] In general, motifs that are derived from highly conserved
sequences, are not desirable in making a combinatorial ribozyme
library, as the sequence would be present in every potential
target. By highly conserved is meant that all amino acids found in
a contiguous sequence of amino acids found in a motif are
identical. An optimal situation is where several conserved sequence
possibilities exist, all of which can contribute to a conserved
motif. By conserved is meant that amino acid sequences in a motif
are at least 80% and more preferred at least 90% identical. This
increases the target specificity of the combinatorial ribozyme
pool. In this case, individual ribozymes contained within the
library specifically target the production of functionally unique
molecules. Ribozymes can be designed to motifs of any length. As
the length of a motif increases, different ribozymes can be
targeted to nucleotides encoding contiguous conserved or partially
conserved amino acid sequences throughout the length of the motif.
Generally, a combinatorial ribozyme library is designed to target
an RNA encoding a partially conserved amino acid sequence found in
a motif of interest. By partially conserved is meant that the amino
acid sequences found in a motif are at least 60% identical.
[0029] When designing the combinatorial ribozyme library, all
combinations of nucleotide sequences that give rise to the chosen
motif based on codon degeneracy and usage and the location of the
ribozyme cleavage sites are taken into consideration. The
target-binding nucleotides of the combinatorial ribozyme library
are therefore degenerate. This insures that the ribozyme library
can target all possible permutations of the targeted sequence. For
expression, both sense and antisense sequences are prepared: the
sense strands are annealed to the corresponding antisense strands
to form double stranded DNA molecules. When transcribed in a host
cell culture, the sense DNA produces RNA which is complementary to
an mRNA sequence encoding a motif of interest and contains a
catalytic domain designed to cleave the mRNA sequence. Each member
of a ribozyme library includes two stretches of antisense
oligonucleotides, each preferably between 5-9 nucleotides (nt) long
and optimally 6 to 8 nucleotides long, to bind to the mRNA, with
the sequence forming the catalytic domain or catalytic core in
between. The bases immediately adjacent to either side of the
catalytic core in the sense strands constitute the ribozyme binding
sequence when expressed as RNA that is complementary to an mRNA
sequence. The mRNA target contains a consensus cleavage site for
the ribozyme. For hammerhead ribozymes the triplet GUC is best but
the sequence NUN (N=any nucleotide) also can be targeted. If the
catalytic is derived from a hairpin ribozyme, the triplet GUC is
also preferred (Kashani-Sabet and Scanlon, (1995) Cancer Gene
Therapy 2:213-223; Perriman, et al., (1992) Gene (Amst.)
113:157-163; Ruffner, et al, (1990) Biochemistry 29:10695-10702);
Birikh, et al., (1997) Eur. J. Biochem. 245:1-16; Perrealt, et al.
(1991) Biochemistry 30:4020-4025). Generally, the entire
ribozyme-mRNA binding sequence is about 10 to 30 nucleotides in
length with 11-17 nucleotides being preferred. The catalytic region
generally is about 22 nucleotides in length (Uhlenbeck et al.
Nature 328:596-603).
[0030] The oligonucleotides for the sense and antisense DNA strands
can be simultaneously synthesized on solid supports in a matrix
format, and simultaneously deprotected and cleaved. If
complementary pairs of oligonucleotides are simultaneously
synthesized, deprotected and cleaved in a matrix format, they can
be simultaneously annealed and ligated to a vector. Another method
of producing these constructs is to make shorter oligonucleotides
with shorter complementary regions. Each partially complementary
oligonucleotide, each having one part out of the two parts of the
targeted motif and a restriction enzyme site, is annealed, extended
using a DNA polymerase, and digested with the appropriate
restriction enzymes prior to ligation. For example, when targeting
a zinc-finger motif, Cysteine-X-X-Cysteine, where X is any other
amino acid, the targeted sequence on the mRNA is
5'-NUGYNNNNNNUGY-3' (SEQ ID NO:28) where N is any base and Y is U
or C (see Example 1). The ribozyme pool would need to contain the
target sequence 5'-RCANNNNNRCA (SEQ ID NO:29) (R=A or G) in the
target binding region. An example is as follows (SEQ ID NO:30-31)
(Y=C or T):5 'GGAATTCRCANNNCTGATGAGTCCGTGAGACTCAGGCACTC-
CTGCTTTNYGTNCCTAGG5' Bold indicates the nucleotides encoding the
ribozyme catalytic domain and the underlined sequences encode the
sites that bind to the complementary mRNA encoding the zinc-finger
motif. After annealing, both strands of the oligonucleotide are
extended using a DNA polymerase and then restriction enzyme
digested, in this example, with EcoR I and BamH I (italicized bases
in upper and lower strands, respectively). The length of the
complementary region can be longer or shorter depending on the
annealing conditions. Extra bases can be added at the 5' ends of
both strands to improve cutting by the restriction enzymes.
Alternatively, a single oligonucleotide is annealed to the delivery
vector, ligated and the complementary strand can be filled in by a
DNA polymerase or the complementary strand can be filled in before
ligation. Three oligonucleotides can be annealed together with the
delivery vector; intervening gaps are filled in by a DNA polymerase
and ligated with a DNA ligase.
[0031] For annealing the complementary oligonucleotide DNA strands
encoding the combinatorial ribozyme library, special conditions are
not required. For example, both strands can be dissolved in water
then mixed at about a one to one molar ratio. They can be mixed in
almost any buffer system, T4 ligase buffer, Exonuclease 3 buffer,
Mung Bean Nuclease buffer. No special heating is required, room
temperature is adequate. This mixing and annealing of the
oligonucleotide strands generally occurs in multiwell microtiter
dishes although other appropriate apparatus also can be used. A
means for determining directionality of expression can be included
in the oligonucleotide DNA. Conveniently the means is the use of
unique non-blunt end forming restriction enzyme sites at both ends
of the oligonucleotide DNA, so that the two oligonucleotides to be
annealed share complementary sequences except at the ends where
they will be able to bind to a restriction enzyme site. For the
restriction enzyme sites, any non-blunt end forming restriction
enzyme site can be used at either end, depending on the sites
within the DNA vector into which the oligonucleotide DNA fragment
is to be ligated. Having different sites at each end provides
directionality for ligation. Any restriction enzyme that produces
unique non-blunt cohesive ends suitable for ligation by DNA ligase
can be used, for example Aat II, EcoR I, BamH I, Hind III, Pst I.
If necessary, a restriction enzyme site with a cohesive end can be
used with a restriction enzyme site that produces a blunt end.
Alternatively, the two oligonucleotides can be completely
complementary, including the ends and digested with restriction
enzymes prior to ligation with the delivery vector. In this case,
it is preferred that the restriction enzyme sites do not occur
within the oligonucleotide DNA encoding the ribozyme, otherwise
partial digestions will be required. Restriction enzyme digestion
is routinely performed using commercially available reagents
according to the manufacturer's recommendations and will vary
according to the restriction enzymes chosen.
[0032] The members of the combinatorial ribozyme library are
introduced into any of a variety of vectors, depending on the
availability of restriction enzyme sites, intracellular location,
and transcriptional regulatory elements for delivery and expression
of the ribozymes in the target host cell. The delivery vector into
which the ribozymes are to be ligated is digested with the
appropriate restriction enzymes, either simultaneously or
sequentially, to produce the appropriate ends for directional
cloning of the oligonucleotides. The oligonucleotide DNAs
preferably contain compatible ends to facilitate ligation to the
vector in the correct orientation. For synthetic oligonucleotide
ligation, the ends compatible with the vector can be designed into
the oligonucleotides. Alternatively, the compatible ends can be
formed by restriction enzyme digestion of the ligation of linkers
to the oligonucleotides containing the appropriate restriction
enzyme sites. The vector also can be modified by the use of
linkers. The restriction enzyme sites are chosen so that
transcription of the cloned oligonucleotides from the vector
produces a ribozyme targeted to the mRNA transcribed from a target
nucleic acid that encodes a motif of interest.
[0033] The vector encoding the ribozyme or ribozyme library also
may encode a marker protein. The marker protein is used for
selection of cells that have been transfected/infected with the
library-encoding vector. The marker may provide antibiotic
resistance. The marker also may provide for visual selection (for
example .beta.-galactosidase or green fluorescent protein). The
marker can also be a transmembrane protein (for example CD4).
[0034] Once digested, the vector and oligonucleotides can be
purified by gel electrophoresis, chromatography or
phenol/chloroform extraction and ethanol precipitation. The optimal
purification method depends on the size and type of the vector and
oligonucleotides, however, both can be used without purification.
Generally, the oligonucleotide DNA does not contain 5'-phosphate
groups and, therefore, the phosphate groups on the vector produced
by restriction enzyme digestion are necessary for
oligonucleotide-vector ligation. The 5'-phosphate groups can be
added to the oligonucleotides by chemical or enzymatic means before
or after annealing and the 5'-phosphate can be removed from the
digested vector to prevent vector-vector ligation. For ligation,
ratios of oligonucleotide DNA to vector DNA range from
approximately 4:1 to 6:1. The ligation reaction is performed using
T4 DNA ligase or any other enzyme that catalyzes double stranded
DNA ligation. Reaction times and temperature can vary from 5
minutes to 18 hours to from room temperature to 15.degree. C. The
delivery vector containing the combinatorial ribozyme library
optionally is treated to increase the supercoiling of the delivery
vector DNA, for example using DNA gyrase so as to improve uptake of
the DNA into a recipient cell, such as a packaging cell or the
intended target host cell.
[0035] One method for expression of the ribozyme library employs
recombinant retroviruses. These vectors generally include as
operatively linked components, retroviral long terminal repeats,
packaging sequences and cloning site(s) for insertion of
heterologous sequences. Other operatively linked components can
include a nonretroviral promoter/enhancer and a selectable marker
gene. Examples of retrovirus expression vectors which can be used
include DC-T5T (Sullenger et al. 1990. Mol Cell Biol.
10:6512-65230, kat (Blood. 1994 83:43-50), BOSC (Proc. Natl. Acad.
Sci. (USA) (1993) 90:8392-8396), pBabe (Proc. Natl. Acad. Sci.
(USA) (1995) 92:9146-9150) and RetroXpress.TM. (Clontech, Palo
Alto, Calif.).
[0036] In some instances, it is desirable to increase expression of
the ribozyme library utilizing other promoters and/or enhancers in
place of the promoter and/or enhancers provided in the expression
vector. These promoters in combination with enhancers can be
constitutive, tissue specific or regulatable. Any promoter/enhancer
system functional in the target host cell can be used. (See for
example, Molecular Virology pp. 176-177; Hofman, et al. 1996. Proc.
Natl. Acad. Sci. (USA) 93:5185-5190; Coffin and Varmus, 1996.
Retroviruses. Cold Spring Harbor Press, NY; Ausubel et al. 1994.
Current Protocols in Molecular Biology. Greene Publishing
Associates, Inc. & Wiley and Sons, Inc.). Examples include: CMV
immediate-early promoter, SV40 promoter, thymidine kinase promoter,
metallothionein promoter, and the tetracycline operator (Hoffman et
al., (1996) Proc. Natl. Acad. Sci (USA) 93:5185-5190. Other methods
to obtain recombinant retrovirus particles also can be used. For
example, the oligonucleotide DNAs are functionally linked to
eukaryotic transcriptional elements and are flanked by a retroviral
packaging signal and 5' and 3' LTRs. This entire retrovirus
construct is functionally linked to the T7 RNA polymerase promoter
and T7 terminator. Also encoded by the vector but not within the
retroviral construct is a gene functionally linked to a eukaryotic
promoter that expresses a T7 RNA polymerase (T7pol) that contains a
nuclear localization signal (T7pol-nls). Following transfection of
this vector into a retroviral packaging cell, the T7-nls is
expressed and localized in the nucleus where it transcribes
recombinant retroviral genomes that are packaged by the retroviral
genes expressed by the packaging cell. Because of the high
transcriptional activity of T7pol-nls, high recombinant retrovirus
titers can be achieved. Similar vectors, utilizing other
DNA-dependent RNA polymerases, such as, SP6 or T3 also can be
used.
[0037] To package the recombinant retrovirus vectors containing the
ribozyme library, cell lines are used that provide in trans the
gene functions deleted from the recombinant retrovirus vector such
that the vector is replicated and packaged into virus particles.
The genes expressed in trans encode viral structural proteins and
enzymes for packaging the vector and carrying out essential
functions required for the vector's expression following infection
of the target host cell. Packaging cell lines and retrovirus vector
combinations that minimize homologous recombination between the
vector and the genes expressed in trans are preferred to avoid the
generation of replication competent retrovirus. Packaging systems
that provide essential gene functions in trans from co-transfected
expression vectors can be used, as can packaging systems that
produce replication competent retroviruses. Following packaging,
the recombinant retrovirus is used to infect target cells of
interest. The envelope proteins expressed permit infection of the
target cell by the recombinant retrovirus particle. Retrovirus
packaging cell lines which can be used include BOSC23 (Proc. Natl.
Acad. Sci. (USA) 90:8392-8396), PT67 (Miller and Miller. 1994. J.
Virol. 68:8270-8276, Miller. 1996. Proc. Natl. Acad. Sci. (USA)
93:11407-11413), PA317 (Mol. Cell Biol. 6:2895 (1986)), PG13, 293
cells transfected with pIK6.1 packaging plasmids (U.S. Pat. No.
5,686,279), GP+envAM12 (Virology 167:400 (1988), PE502 cells
(BioTechniques 7:980-990 (1989)), GP+86 (Markowitz, et al. 1988. J.
Virol. 62:1120-1124) .PSI.-Cre (Danos and Mulligan. 1988. Proc.
Natl. Acad. Sci. (USA) 85:6460-6464). The preferred titer of
recombinant retrovirus particles is about 10.sup.5-10.sup.7
infectious particles per millimeter. If these titers cannot be
achieved the virus also can be concentrated before use.
[0038] In addition to recombinant retrovirus systems, other viral
packaging systems such as adenovirus-associated virus (AAV),
adenovirus, Sindbis virus, Semliki Forest virus, Epstein Barr
virus, herpes simplex virus, HIV, or vaccinia virus can be used.
Each of these systems has a different host range and can be used to
infect cells that are refractory to retrovirus expression (i.e.,
non-dividing cells). In the Sindbis virus system (Invitrogen, San
Diego, Calif.), the oligonucleotides to be expressed are ligated
into the multiple cloning site of a Sindbis virus DNA vector, e.g.
pSinRep5, operatively linked to a Sindbis subgenomic promoter and
polyadenylation site; the oligonucleotides replace the Sindbis
virus structural protein genes. pSinRep5 includes an SP6 RNA
polymerase promoter for the in vitro synthesis of recombinant
Sindbis virus genomes; a packaging signal for recombinant RNA
packaging; and the Sindbis nonstructural polyprotein gene open
reading frame. For the production of Sindbis virus particles, the
recombinant Sindbis vector encoding the oligonucleotide DNA is
linearized, transcribed into RNA and co-transfected into vertebrate
(BHK-21, Vero) or invertebrate cells (Drosophila) with RNA
transcribed from the helper vector, pDH-BB, that encodes the viral
structural proteins. Following transfection, the recombinant
Sindbis genomic RNA acts as a mRNA, is translated into the Sindbis
virus polymerase, and expresses the encoded ribozyme and the
structural proteins from the helper RNA. Because of Sindbis virus'
wide host range, the recombinant Sindbis can be packaged and used
to express the ribozyme library in mammalian, avian, reptilian,
insect cells (e.g., mosquito and Drosophilla cells). See for
example, Xong, C. et al. (1989) Science 243:1188-1191; Huan, H. V.
et al. (1993) U.S. Pat. No. 5,217,879; Hahn C. S. et al. (1992)
Proc. Natl. Acad. Sci. (USA) 89:2679-2683; Huang, M. and Sommers,
J. (1991) J. Virol. 65:5435-5439.
[0039] For ribozyme expression in AAV, the oligonucleotide DNA is
closed into an AAV expression vector, such as ALAPSN, that contains
a cloning site functionally linked to a promoter such as a Moloney
leukemia virus promoter and flanked by AAV terminal repeats and a
packaging signal, a means for selection. As an example, ALAPSN
comprises a neomycin resistance gene functionally linked to SV40
transcription control elements. Similar AAV vectors, such as CWRSP
and CWRSP.N., with comparable features also can be used. As an
example, to produce recombinant AAV particles, 293 cells are
infected with adenovirus type 5; then the infected cells are
co-transfected with an ALAPSN plasmid-oligonucleotide DNA construct
and an AAV helper plasmid, e.g. pAAV/Ad (Samulski et al., (1989) J.
Virol. 63:3822-3828). As recombinant AAV is produced, the 293 cells
undergo cytopathology, becoming spherical and lose their ability to
adhere to a tissue culture surface. Following development of
maximal cytopathology the supernatant and/or cell lysate is
harvested and, if necessary, concentrated (Halbert et al. 1997. J.
Virol. 71:5932-5941). Other methods for producing recombinant AAV
also can be used, for example as described in U.S. Pat. No.
5,354,678. The combinatorial ribozyme library also can be expressed
using adenovirus expression systems as described in U.S. Pat. No.
5,631,236, U.S. Pat. No. 5,670,488, WO94/28152, WO95/14091,
EP0707071, WO96/05321, WO95/14101, WO95/14102, WO97/00326,
EP94202322.7.
[0040] For vaccinia virus expression, a replication competent
vaccinia virus can be used. The oligonucleotides to be expressed
are operatively linked to a vaccinia virus promoter, for example,
P11. In a preferred embodiment, vaccinia virus strain MVA is used
because it expresses recombinant genes but contains a deletion that
renders it replication incompetent in mammalian cells. Therefore,
nucleic acids can be expressed in target host mammalian cells
without the development of vaccinia virus induced cytopathology.
The recombinant vaccinia virus strain MVA is produced by infecting
chicken embryo fibroblasts (CEF) with vaccinia MVA and transfecting
the transfer vector, pG01, into which has been ligated the ribozyme
and a marker gene (beta galactosidase) functionally linked to a
vaccinia promoter, such as P11, and flanked by the MVA genome
sequences that flank the site of the MVA genomic deletion. The
P11-ribozyme/beta-galactosidose construct is inserted into the MVA
genome by homologous recombination. Recombinant viruses can be
identified by in situ staining for beta-galactosidase expression
with X-gal (Wyatt et al. (1995) Virology 210:202-205).
[0041] The combinatorial ribozyme library also can be expressed
from plasmid expression vectors that are transfected directly into
target host cells, including mammalian cells, although an
intervening bacterial amplification step can be performed prior to
delivery of the library to the target host cells. The direct
delivery of the plasmid expression vector into the target host
cells without an intervening bacterial cloning or transformation
step is preferred because it provides a significant savings in time
and expense and increases the number of genes and ribozyme
libraries that can be studied. Expression plasmids contain cloning
sites operatively linked to transcriptional regulatory elements
functional in the target host cells. When the target host cells are
mammalian cells, examples of transcriptional regulatory elements
that can be used include SV40, CMV, metallothionein, or
tetracycline transcriptional regulatory elements: pCEP4
(Invitrogen, San Diego, Calif.), pCMVb, (Clontech, Palo Alto,
Calif.), pAlter-MAX (Promega, Madison, Wis.). The plasmid
preferably contains sequences to provide high-copy episomal
replication and selectable markers for stable maintenance of the
vector in the host cell. The plasmids containing the
oligonucleotide DNA are transfected directly into the target cell
of interest. To increase transfection efficiency, preferably the
plasmids are supercoiled with a gyrase. The oligonucleotides
encoding the combinatorial ribozyme library alternatively can be
ligated into plasmids and functionally linked to the T7, SP6, T3 or
a similar RNA polymerase promoter. The plasmid expression vectors
that can be used include pGEM-3Z and pAlter-Exl (Promega, Madison,
Wis.). The plasmid-oligonucleotide DNA construct is transfected
into mammalian cells that are infected with a vaccinia strain such
as MVA that expresses the appropriate RNA polymerase (Wyatt et al.,
(1995) Virology 210:202-205). For the example of vaccinia MVA T7,
the T7pol transcribes the oligonucleotides from the plasmid vector.
The vaccinia MVA amplifies the plasmid-oligonucleotide DNA
construct copy number, resulting in an increased intracellular
template concentration for T7pol transcomplementation and increased
ribozyme expression and thus activity.
[0042] Other systems for the expression of nucleic acids
functionally linked to T7 RNA polymerase or other bacteriophage
promoters (SP6 or T3) also can be used. Ribozyme expression can be
performed with a recombinant retrovirus vector containing the
oligonucleotides encoding the ribozyme functionally linked to a T7
RNA polymerase promoter (T7pro) and T7 terminator. This expression
cassette is flanked by 5' and 3' LTRs, a packaging signal and
includes the T7pol gene, that encodes a T7pol that contains a
nuclear localization signal (T7pol-nls), functionally linked to a
eukaryotic promoter. In this system, the expressed T7 protein is
transported to the nucleus for transcription. Due to the high
transcriptional activity of T7pol, high intracellular levels of
ribozyme can be achieved. Optionally, the ribozyme can be fused to
second ribozyme that acts intramolecularly to free the ribozyme
targeting the mRNA of interest.
[0043] Transfection of nucleic acid (DNA or RNA) encoding the
combinatorial ribozyme library into cells is required for either
packaging of recombinant vectors into virus particles or direct
transfection of plasmids that express the combinatorial ribozyme
library into target host cells can be mediated by a variety of
chemicals including calcium-phosphate, polybrene, DEAE-dextran, and
liposomes. The calcium-phosphate method includes incubating the
target cell with a calcium phosphate-nucleic acid co-precipitate.
Polycations such as polybrene (about 4-10 micrograms/ml), a
polycation that acts by neutralizing the net negative surface
charges on the virus and cells (Stoker. "Retroviral Vectors" In
Molecular Virology: A Practical Approach, Davison and Elliott,
eds., p 187) or DOSPER (Boehringer-Mannheim) also can be used to
increase the efficiency of transfection of low molecular weight
DNA. Liposomes are available from a variety of commercial suppliers
and include DOTAP.TM. (Boehringer-Mannheim), Tfx.TM.-50,
Transfectam.RTM., ProFection.TM. (Promega, Madison Wis.), and
LipofectAmin.TM., Lipofectin.RTM., LipofectAce.TM. (GibcoBRL,
Gaithersburg, Md.). In solution, the lipids form vesicles that
associate with the nucleic acid and facilitate its transfer into
cells by fusion of the vesicles with cell membranes or by
endocytosis. Alternatively, DNA can be introduced into cells by
electroporation. Each of these systems differ in their transfection
efficiency for a given cell line. If transfection conditions for a
given cell line have not been established or are unknown, they can
be determined empirically (Maniatis supra). The number of ribozymes
expressed per cell depends on the multiplicity of infection for a
virus vector or the amount of DNA transfected per cell for a
plasmid vector.
[0044] From one to multiple ribozymes in a chosen delivery vector
are introduced into the recipient cell. When a retrovirus vector is
used, following preparation of recombinant retrovirus in a
packaging cell, the recombinant retrovirus is used to infect a host
cell containing mRNA transcribed from a target nucleic acid
encoding the motif of interest. The infected or transfected host
cell is grown and the phenotype of the infected or transfected host
cell is analyzed to determine any alterations in phenotype as
compared to an uninfected or untransfected host cell. Optionally,
infected or transfected cells are isolated or selected from the
population of normal cells. Alterations in cell phenotype are then
correlated so as to assign a function to a product coded for by the
cleaved mRNA. DNA encoding the ribozyme expressed in the host cell
can be isolated and sequenced to identify the sequence of the
target mRNA, the gene from which it is transcribed and the encoded
protein. This can be done, for example, by PCRing the ribozyme
encoding sequence for example, from cellular DNA, or reverse
transcription-PCR of RNA, then sequencing the ribozyme encoding
sequence from the viral or plasmid expression vector in the target
cell.
[0045] The target host cell can be any cell of interest that
expresses a disease associated phenotype or a phenotype that can be
differentiated from a "normal" or control cell. To determine if a
target nucleic acid encoding a motif of interest is required for
the cellular phenotype, a ribozyme library designed to cleave the
transcription product of the target nucleic acid is constructed and
expressed in the target host cell(s) that are then assayed for an
altered cell phenotype. The altered phenotype can be any phenotype
which can be detected, for example modified cell growth, DNA
synthesis, synthesis of a protein(s), chemical responsiveness,
apoptosis, morphologic changes, cell viability, replication,
differentiation, expression of biologically active compounds (e.g.,
steroids), proliferation, drug susceptibility, the expression of
cell surface molecules such as receptor molecules and antigens.
Proteins that regulate gene expression in cells also can be
identified. For example, this can be accomplished by monitoring the
expression of a reporter gene expressed from a promoter that is
active in, for example, tumor cells in the presence of ribozymes
targeted to an mRNA encoding a motif known to function as DNA
binding protein. Conversely, for the identification of proteins
responsible for the maintenance of a normal cell phenotype,
ribozymes can be constructed to mRNAs encoding proteins that
contain a motif of interest and which are expressed in the normal
cells which are then assayed for an altered phenotype. In either
approach, proteins and genes associated with disease pathways or
phenotypes can be identified.
[0046] In order to evaluate an alteration in cell phenotype, any of
a variety of methods can be used depending at least in part on the
phenotype of interest and the function associated with the targeted
motif. In host cells which amplify and express ribozymes,
phenotypic change can be monitored directly. For example, if the
function of a protein containing the targeted motif prevents
apoptosis and it is inhibited by one or members of a combinatorial
ribozyme library, the host cell undergoes specific types of
morphologic changes, such as nuclear condensation and DNA
fragmentation, following ribozyme expression. If the targeted motif
is found in a protein that is involved in drug susceptibility, this
function can be identified by monitoring cells for altered
resistance or susceptibility to the particular drug or drugs.
[0047] Combinatorial ribozymes also can be used for functionating
cellular and viral motif containing genes that are involved in
virus replication. Combinatorial ribozyme libraries can be targeted
to mRNAs encoding protein motifs that are postulated to be involved
in a virus' lifecycle. If the targeted motif is found in a protein
that affected replication, virus titers or cytophatic effects may
increase or decrease. Various stains can be employed to determine
whether the function of the targeted nucleic acid affects for
example, cell viability or membrane permeability. If the targeted
nucleic acid encoding the motif of interest affects cell cycle
regulation and transformation this can be monitored by measuring
the incorporation of a labeled nucleotide into the cell.
Antibody-based assays can be employed to detect the presence or
absence of a protein of interest such as a cell membrane receptor.
Additional types of assays known to those of skill in the art can
be employed depending on the phenotype or cellular property that is
being analyzed.
[0048] Phenotypic change also can be monitored, for example, by
evaluating ribozyme activity by comparing the targeted mRNA levels
in cells expressing and cells that do not express one or more
members of the combinatorial ribozyme library. Total cellular or
cytoplasmic RNA can be purified by a variety of methods (Maniatis
supra pp. 7.6-7.29) and analyzed by Northern or dot blot (Maniatis
supra pp. 7.37-7.57). mRNA can be assayed by reverse
transcription-PCR employing primers that flank the targeted
cleavage site(s). The absence or decreased production of a PCR
product is entirely indicative of ribozyme indicative of ribozyme
activity (Baier et al. 1994. Molecular Immunology 31:923-932).
[0049] The methods and compositions of the subject invention can be
used to identify the function of nucleic acids encoding proteins
containing motifs of interest. Motif-directed ribozyme libraries
can be designed and constructed to target virtually any sequence
encoding a motif for which a conserved or nearly conserved sequence
can be identified. Conserved sequences have been described for
paspases and protein kinases. In addition many other classes of
enzymes can similarly be targeted. A conserved sequence encoding a
zinc-finger domain that is found in many proteins has been
described. Similarly, a sequence conserved in integrins has been
described. Therefore, it is possible to target motifs in numerous
proteins including enzymes, adhesion molecules, signaling molecules
and structural proteins having a variety of physiological functions
including enzyme activity, protein synthesis, biological factor
expression or regulatory effector function, which alter various
cellular phenotypes or responses including changes in cellular
proliferation kinetics, changes in cellular viability, resistance
to facilitated cell death, resistance to antibiotics, magnetic
separation, directed migration, and preferential adhesion.
[0050] Following the identification of cells that exhibit an
altered cellular phenotype in response to expression of a ribozyme,
the host cell having an altered cellular phenotype is isolated or
selected for on the basis of expression of an appropriate marker,
which can be for example, a cell surface molecule, a drug
resistance protein, an enzyme, or a bioluminescent molecule. Cells
can also be isolated using FACS sorting, magnetic separation
techniques, drug selection, visual selection, or methods based on
enzymatic activity.
[0051] DNA or RNA is isolated from the host cell by standard
molecular biology techniques and can be PCR amplified for
sequencing as an initial step towards characterization of the
corresponding gene, transcription product and protein. For example,
this can be done by PCR amplifying the ribozyme-coding region of
the viral or plasmid vector that delivered the ribozyme to the
cell. The primer pair used to amplify the ribozyme sequence is
derived from the vector sequences flanking the ribozyme insert. The
PCR product is then sequenced to determine the mRNA sequence
targeted by the ribozyme(s). Based on this information, the entire
gene sequence can be determined and the sequence of the encoded
protein can be deduced. PCR products also can be cloned into
vectors for further analysis, or used as probes for identification
of target nucleic acids.
[0052] In the following examples, a combinatorial ribozyme library
is targeted to a zinc finger motif, C-X-X-C. Included in the
library, at an equivalent proportion to the other components, is a
ribozyme targeting green fluorescent protein (GFP). Green
fluorescent protein (GFP) from the firefly Aequorea victoria emits
bright green light upon exposure to UV light without the
requirement of additional proteins, substrates, or cofactors. EGFP
encodes a protein that has a single, red shifted spectrum and
increased expression relative to GFP, and therefore, is easily
monitored in living cells by fluorescence microscopy and
fluorescence-activated cell sorting (FACS).
[0053] To demonstrate that the combinatorial ribozyme library can
be used to inactivate genes involved in a given phenotype, the
library targeting the zinc finger motif and containing the
EGFP-targeted ribozyme is introduced into CHO-AA8 Tet-Off cells or
293 Tet-Off cells (Clontech, Palo Alto, Calif.) that express EGFP.
Ribozyme activity is inversely proportional to reporter gene
signal. Alternatively, the zinc finger motif can be incorporated
into the EGFP sequence, expressed in either cell type described
above, and inactivated by the combinatorial ribozyme library. Other
reporter genes, for example chloramphenicol acetyltransferase
(CAT), beta-galactosidase, or alkaline phosphatase, also can be
used.
[0054] Kits containing combinatorial anti-motif ribozyme libraries
also are provided. The containers of kit can contain a
combinatorial library directed to motifs either as individual
members of the library, or as a complete library. Optimally the kit
contains vectors including plasmid vectors, retrovirus expression
vectors and adeno-associated virus expression vectors for cloning
the library. Additional components of the kit can include
antibodies for recognition of a marker protein and PCR primers for
amplification of the nucleotides encoding the ribozymes.
[0055] The following examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
Example 1
Design of Combinatorial Ribozyme Library to a Zinc Finger Motif
[0056] The following example discloses methods to design double
stranded DNA oligonucleotides that code for a combinatorial
hammerhead ribozyme library targeted to the zinc finger motif,
C-X-X-C (X=any amino acid). Hammerhead ribozymes contain two
recognition domains that are complementary to the mRNA encoding the
motif of interest. Each recognition domain is composed of at least
6 nucleotides flanking both ends of the catalytic core. The optimum
cleavage site of the target mRNA is a U. Based on these
considerations the minimal target sequence contained in the coding
sequence of the motif and cleavable by a ribozyme is determined.
Also, considered is the known degeneracy of the genetic code.
Mammalian codon usage may also be considered. Thus, the zinc finger
motif, described above with the amino acid sequence, C-X-X-C, the
sequence is first reverse translated to: 5'-UGY-NNN-NNN-UGY (SEQ ID
NO:32) (N=any nucleotide, Y=C or U). The amino acid sequence is
scanned for amino acids that are preferably found at or near the
middle of the motif that are coded for by codons that contain a U
as a fixed position. In this case none are available, therefore, to
target a ribozyme library to this sequence requires fixing one of
the variable residues as a U and extending the recognition sequence
arms of the ribozyme 5' and 3' from this position to include the
less or invariable elements characteristic of the sequence. Taking
into consideration that the hammerhead ribozyme contains two
recognition sequences each comprised of 6 nucleotides, the minimal
recognition sequence of the zinc motif is: 5'-NUGYNUNNNNUGY (SEQ ID
NO:33) wherein NUN=cleavage recognition site, with cleavage
occurring 3' to the underlined nucleotide. The underlined
nucleotide is not targeted by the ribozyme because it does not
hybridize or anneal with the binding sequences of the ribozyme. The
structure of the ribozyme annealed to the target sequence is shown
in FIG. 1.
[0057] The number of ribozymes targeting the minimum sequence is
calculated by multiplying the number of nucleotides that may occupy
each position of the binding regions. Omitted from this calculation
is the nucleotide 5' to the cleavage site (underlined nucleotide)
because it is not part of the ribozyme binding region. For the
above example, the number of ribozymes to be made equals:
4.times.1.times.1.times.2.times.4.-
times.1.times.4.times.4.times.4.times.1.times.1.times.2=4096.
[0058] Oligonucleotides encoding the combinatorial ribozyme library
to the zinc finger motif are made in a 96-well matrix using
parallel array technology and annealed to form double stranded DNA
with unique Hind III and Cla I sites at each of the 5' and 3' ends,
respectively, for ligation into a retrovirus vector. The general
structure of two complementary oligonucleotides of the
combinatorial library is as follows, with the catalytic core of a
hammer head ribozyme in bold. The underlined regions are the
ribozyme binding sequences when expressed as RNA that are
complementary to all possible permutations of a mRNA sequence
encoding the zinc finger motif (SEQ ID NO:34-35).
1 5' AGCTTRCANNNCTGATGAGTCCGTGAGGACGAAANRCANAT 3' 3'
AYGTNNNGACTACTCAGGCACTCCTGCTTTNYGTNTAGC 5'
[0059] For annealing, approximately 1.0 microgram of each
complementary oligonucleotide is dissolved in water and mixed at a
one to one molar ratio in a 96-well microtiter plate at room
temperature. The 5' end (left end) of the double stranded DNA
fragment overlaps with an Hind III restriction enzyme site. The 3'
end of the fragment (right end) overlaps with a Cla I site.
Example 2
Preparation of a Family of Retrovirus Plasmid Vectors
[0060] The purpose of this experiment is to prepare a retrovirus
plasmid vector library containing the double stranded
oligonucleotide DNA encoding the combinatorial ribozyme library to
the zinc finger motif described in Example 1, supra. pLNCX, (50
micrograms, Clontech, Palo Alto, Calif.), which contains an
extended viral packaging signal, multiple cloning site and neomycin
resistance gene flanked by the Moloney murine leukemia virus 5' and
3' long terminal repeats and an ampicillin resistance gene is
digested with restriction enzymes, Hind III and Cla I.
Approximately, 0.5 to 2 .mu.g of digested plasmid is placed into a
well of a multi-well (e.g. 96 well) plate. The library of annealed
oligonucleotides prepared in Example 1, supra, are added
individually at 4 to 6 fold excess of the Hind III/Cla I treated
pLNCX. The oligonucleotide DNA is ligated into pLNCX by adding a
tenth volume of 10.times. T4 DNA ligation buffer and T4 DNA ligase.
The final concentration of the ligation buffer components and T4
DNA ligase are: 0.05 M Tris-HC1 (pH 7.6), 10 mM MgCl.sub.2, 10 mM
dithiothreitol (DTT), 50 .mu.g/ml bovine serum albumin (Fraction V;
optional), 1.0 mM ATP, 0.05 Weiss units of bacteriophage T4 DNA
ligase per microliter. The ligation is performed for 4-8 hours at
16.degree. C.
[0061] Also prepared, is a retrovirus vector encoding a known,
active anti-EGFP ribozyme following the identical protocol.
Example 3
Transfection of Mammalian Cells Using Retrovirus Plasmid
Vectors
[0062] The purpose of this experiment is to package the retrovirus
plasmid vector library prepared in Example 2, supra, and an
identical retrovirus plasmid containing a specific anti-EGFP
ribozyme in retrovirus particles. Using a calcium phosphate
precipitation method (Keck, et al. (1990) Cell 61:801-809) Cochran,
et al. (1985) Proc. Natl. Acad. Sci. (USA) 82:19-23) for
transfecting DNA into mammalian cells, 0.1 to 0.5 micrograms of
ligated plasmid/oligo DNAs from Example 2, supra, is transfected
into approximately 1,0000 to 25,000 PT67 cells per well of a
96-well plate cultured in 100 .mu.l of minimal essential media
(MEM) supplemented with 10% fetal calf serum (FCS). Four hours
later the media is replaced with fresh MEM supplemented with 10%
FCS and incubated at 37.degree. C. for 48 hours. The retrovirus
vector contains a neomycin resistance gene; therefore, G418
selection is used to obtain a population of cells that stably
express the transfected vector and to monitor virus titers.
Recombinant retrovirus production is monitored by titering aliquots
of the transfected cell supernatant in a focus forming assay in
which cells infected with the recombinant retrovirus become
resistant to G418 (Clontech, Palo Alto, Calif.). When virus titers
>10.sup.5/ml are reached, usually between 2-7 days, the viruses
are harvested, and random pools of retroviruses containing the
combinatorial library and the specific EGFP ribozyme are made.
These pools are used to infect into target cells, CHO-AA8-Tet-Off
cells (Clontech, Palo Alto, Calif.), expressing EGFP (CHO-EGFP)
seeded into 96-well plates. Alternatively, the packaged retrovirus
library is titered and infected into fresh PT67 cells at a
multiplicity of 1. These cells are counted then added to multi-well
dishes seeded with target cells. The ratio of retrovirus producing
PT67 cells and target cells can vary from about 1 to 10. As the
retrovirus particles emerge from the PT67 cells, the CHO-EGFP cells
are infected.
Example 4
Analysis of Ribozyme Activity
[0063] This experiment is designed to demonstrate that a ribozyme
in a combinatorial library can inactivate its mRNA resulting in an
altered phenotype of the target host cell. In this experiment,
ribozyme activity is inversely proportional to EGFP expression. The
ribozyme hybridizes to and cleaves the EGFP mRNA, thereby, reducing
EGFP protein expression.
[0064] CHO-EGFP cells (Clontech, Palo Alto, Calif.) are cultured to
near confluency or approximately 50,00-75,000 cells per well of a
96-well plate in MEM with 10% FCS. The recombinant retrovirus
library containing the retrovirus encoding the anti-GFP ribozyme
from Example 3, supra, are randomly pooled and used to infect the
cultures of CHO-EGFP cells. The multiplicity of infection (virus
particle per cell ratio) is about 5-10 to insure that every cell
per well is infected with at least one virus particle. Infection of
target cells is enhanced with polybrene (generally 10
micrograms/ml). Mock infected cells or parental CHO cells that do
not express EGFP serve as controls. Following infection, the cells
are incubated for 48 hours at 37.degree. C. and then assayed for
EGFP expression.
[0065] EGFP expression is assayed by EGFP fluorescence using an
incident light at 488 nm and measuring the emitted light at 507 nm.
The emitted or observed light is detected using the appropriate set
of filters, corresponding to the wavelength of the incident and
emitted light with a Wallac-Victor Flurometer or by a Florescence
Activated Cell Sorter (FACS). Cells with decreased EGFP production
were analyzed by PCR to confirm the presence of the EGFP-specific
zone.
Example 5
Preparation of Plasmid Vector for Non-Retroviral Transfection
[0066] This example discloses the construction of plasmid vectors
that express the combinatorial ribozymes. The oligonucleotides
ribozyme library containing the anti-EGFP ribozyme is ligated into
the multiple cloning site of pCEP4 (Invitrogen, San Diego, Calif.)
operatively linked to the CMV promoter and SV40 polydenylation
signal. pCEP4 is an Epstein Barr virus (EBV)-based vector that is
maintained extrachomosomally in primate and canine cell lines.
pCEP4 contains the nuclear antigen, EBNA-1, for high-copy episomal
replication of the plasmid by the EBV origin of replication, oriP,
and the hygromycin resistance gene for stable maintenance of the
vector. In this example, the oligonucleotides are designed to
contain Hind II and BamH I sites to facilitate ligation into the
expression vector. The anti-EGFP ribozyme is synthesized and cloned
as described for the combinatorial ribozyme library but also
containing Hind III and BamH I sites. The synthesis, annealing and
ligation procedures are the same as those described for the
retrovirus vectors in Example 2, supra.
Example 6
Transfection of Mammalian Cells (Non-Retroviral-Mediated
Transfection)
[0067] This experiment demonstrated the delivery of plasmid DNA
encoding the EGFP ribozyme and the combinatorial ribozyme library
to mammalian cells. Random pools of pCEP4 DNAs containing the EGFP
ribozyme and the combinatorial library are introduced into host
cells by calcium-phosphate precipitation (Cochran et al. (1985)
Proc. Natl. Acad. Sci. (USA) 82:19-23, Keck et al. (1990) Cell
61:801-809). The pCEP4 DNA was treated with gyrase (Mizuuchi et al.
(1994) J. Biol. Chem. 259:9199-9201; Bates et al. (1996)
Biochemistry 35:1408-1416) to increase the transfection efficiency.
Gyrase treatment is carried out for 1 hour at 25.degree. C. in 35
mM Tris-HCl (pH 7.5), 24 mM KCl, 4 mM MgCl.sub.2, 1.8 mM
spermidine, 9 microgram/ml tRNA, 5 mM dithiothreitol, 6.5% (w/v)
glycerol, 100 microgram/ml bovine serum albumin, 12 nM gyrase, 1 mM
ATP (Bates et al. 1996 Biochemistry 35:1408-1416). In either case,
by increasing the amount of transfected DNA from the ligation
reaction, more than one ribozyme targeted to a specific sequence is
transfected per cell. This increases the probability that the
target mRNA is inactivated and an altered phenotype is
produced.
Example 7
Analysis of Ribozyme Activity
[0068] This experiment is designed to demonstrate the inactivation
of EGFP expression in target cells transfected with plasmid vectors
that express the combinatorial ribozyme library and the EGFP mRNA.
Ribozyme activity is inversely proportional to EGFP expression. The
ribozyme hybridizes to and cleaves the EGFP mRNA, thereby, reducing
EGFP protein expression. EGFP is assayed as described above and the
presence of the anti-EGFP ribozyme is confirmed according to the
procedure described in Example 4, supra.
[0069] CHO-EGFP cells (Clontech, Palo Alto, Calif.) are cultured to
near confluency in 96-well plates in MEM supplemented with 10% FCS
as described in example 4, supra. The plasmids for expression of
the combinatorial ribozyme library and the anti-EGFP ribozyme from
example 6, supra, are randomly pooled and used to transfect
CHO-EGFP cells. Approximately, 0.1-0.5 micrograms of plasmid DNA
are used to transfect approximately each well of the CHO-EGFP cells
cultured in 96-well microtiter dishes to insure that every cell was
transfected with at least one plasmid. Mock transfected cells or
parental CHO cells serve as controls. Following transfection, the
CHO-EGFP cells are incubated for 48-72 hours. EGFP is assayed as
described in Example 4, supra.
Example 8
Design of a Combinatorial Ribozyme Library to the Receptor Protein
Kinase Motif
[0070] The following example discloses methods to design double
stranded DNA oligonucleotides that code for a combinatorial
hammerhead ribozyme library targeted to the receptor protein kinase
motif, G-X-H-X-N-[LIVM]-V-N-L-L-G-A-C-T (X=any amino acid; [
]=position contains one of the enclosed amino acids). The sequence
is first scanned, for amino acids that are preferably found at or
near the middle of the motif that are coded for by codons that
contain a U as a fixed position. In this particular case the
position containing Leucine (L), Isoleucine (I), Valine (V) or
Methionine (M) is coded for by nucleotides as follows:
2 L:CUA I:AUA V:GUA M:AUG CUC AUC GUC CUG AUU GUG CUU GUU UUA
UUG
[0071] Thus, L, I, V or M could be coded for by a codon of the
sequence: NUN (N=any nucleotide). Hence, a U is fixed in the second
position of this codon and can be used as part of a ribozyme
cleavage site. As described in Example 1, supra, if a fixed U
residue can not be found in the motif, one can be fixed into a
variable position.
[0072] Taking into consideration that the hammerhead ribozyme
contains recognition sequences comprised of 6 nucleotides and codon
degeneracy, the minimal motif target is, X-N-[LIVM]-V-N (SEQ ID
NO:36), which is reverse translated to yield the minimal nucleotide
target for the combinatorial ribozyme library:
5'-N-A-A-Y-N-U-N-G-U-N-A-A-Y (SEQ ID NO:37) wherein N=any
nucleotide; Y=C or U; NUN-ribozyme cleavage site, with cleavage
occurring 3' to the underlined nucleotide. The underlined
nucleotide is not targeted by the ribozyme because it does not
hybridize or anneal with the binding sequences of the ribozyme. The
number of individual ribozymes necessary to target all possible
nucleotide combinations that may be translated into the motif is
calculated as described in Example 1:
4.times.1.times.1.times.2.times.4.times.1.times.1-
.times.1.times.4.times.1.times.1.times.2=256. The general sequence
of the expressed combinatorial ribozyme library is (SEQ ID
NO:38):
[0073] 5'-RUUNACCUGAUGAGUCCGUGAGGACGAAANRUUN (R=G or A). The
underlined regions are the ribozyme binding sequence when expressed
as RNA that are complementary to the mRNA encoding the receptor
protein kinase motif and the bold region is the catalytic core of
the ribozyme. Other ribozyme libraries also can also be targeted to
other regions of this motif using the guidelines described
above.
Example 9
Design of a Combinatorial Ribozyme Library to the Integrin
Motif
[0074] The following example discloses methods to design
oligonucleotides that code for a combinatorial hammerhead ribozyme
library targeted to the integrin motif:
C-X-[GNQ]-X(1,3)-G-X-C-X-C-X(2)-C-X-C (SEQ ID NO:39). Following the
guidelines described in Examples 1 and 8, the G-X-C-X-C (SEQ ID
NO:40) can be easily targeted where the amino acid, C (Cysteine),
is encoded by UGU or UGC and the amino acid, G (Glycine) is encoded
by GGN. Therefore, the following mRNA sequence can be targeted:
5'-GNNNNUGYNNNUG (SEQ ID NO:41). The general sequence of the
oligonucleotides encoding the combinatorial ribozyme are (SEQ ID
NO:42):
[0075] 5'-CANNNRCTGATGAGTCCGTGAGGACGAAAANNNNC. The underlined
regions and bold region are the ribozyme binding sequences and
catalytic core, respectively. The number of ribozymes necessary to
target all possible nucleotide combination is 32,768. Other
ribozyme libraries also can be targeted to other regions of this
motif using the guidelines described above.
Example 10
Isolation of Cells Expressing a Selectable Marker Associated with a
Ribozyme Expressing Construct
[0076] The following experiment demonstrates the ability to isolate
cells expressing a selectable marker associated with a ribozyme
expressing construct from cell cultures transduced with a library
of pooled ribozymes. A pool of ribozymes directed against the lck
gene was synthesized using oligonucleotides encoding a hammerhead
ribozyme catalytic core flanked by nucleotide sequences
complementary to sequences in the lck mRNA. Specific restriction
endonuclease sites were also engineered into the oligonucleotides
to facilitate directional cloning and recovery of ribozyme
sequences. Sense and antisense oligonucleotides were annealed to
form a double stranded DNA which was then ligated in to an AAV
based plasmid vector using T4 ligase in a manner similar to that
described in Example 2. DNA constructs encoding at least 12
different ribozyme molecules cloned into AAV plasmid vectors were
transfected into a virus packaging cell line, and recombinant virus
was harvested from the supernatant in a manner similar to the one
outlined in the Preferred Embodiments (page 16, lines 23-29). The
resulting recombinant virus was then used to infect the Jurkat
T-cell leukemia line. Infected cultures were harvested and stained
with a fluorescently labelled antibody (Pharmingen, San Diego,
Calif.) directed against the selectable (Lyt-2/CD8a). Marker
expression on the cells was then analyzed by flow cytometry. This
analysis revealed a small population of marker bearing cells
present in the population (see FIG. 3A). This population of cells
was isolated using fluorescence activated cells sorting and
expanded in culture using RPMI 1640 tissue culture medium (Life
Technologies, Grand Island, Utah) according to standard cell
culture techniques. Upon re-analysis by the same method, 100% of
the sorted population was found to express the selectable marker
(see FIG. 3B), suggesting that the recombinant ribozyme-expressing
AAV genome had been stably integrated into the host cell genome.
These results demonstrate that cells expressing ribozyme constructs
can be isolated and separated from those that do not and that these
cells can be expanded in culture for further analysis of cell
phenotype or responsiveness.
Example 11
Isolation of a Population of Cells Which Have Lost a Specific
Cellular Response
[0077] This experiment demonstrates that a population of cells that
have lost a specific cellular response can be isolated from a
culture of cells expressing a library of pooled ribozymes. Jurkat
cells expressing the AAV/ribozyme plasmid selectable marker
(lyt-2/CD8a) derived from the experiment described in Example 10
were stimulated through T-cell receptor engagement with anti-CD3
plus anti-CD28 (Pharmingen, San Diego, Calif.) (Weiss et al.,
(1991) Semin. Immunol., 3:313-324; Abraham et al., (1992) Trends
Biochem. Sci., 17:434-438), for 40 hours, and the induction of
CD69, a cell surface activation protein whose expression is
dependent upon Lck protein kinase function (Goldsmith and Weiss,
(1987) Proc. Natl. Acad. Aci. USA, 84:6879-6883; Straus and Weiss,
(1992) Cell, 70:585-593) was analyzed by flow cytometry using a
fluorescently labeled antibody (Pharmingen, San Diego, Calif.).
This analysis showed that a fraction of ribozyme containing cells
had lost the ability to induce the activation related protein
following cell stimulation (see FIG. 4A). This population of
unreactive cells was isolated using fluorescence activated cell
sorting and recultured. Upon re-analysis, an enrichment of the
unresponsive population was observed. These results show that a
population of cells with a specific altered phenotype or response
can be isolated from a culture of cells expressing a library of
pooled ribozymes directed against a specific gene. In this
particular example, unresponsive cells were isolated from the rest
of the population using flow cytometry.
Example 12
Recovery of Ribozome Sequences from Altered Cells Which Express a
Library of Pooled Ribozymes
[0078] This experiment demonstrates that ribozyme sequences can be
recovered from cells which express a library of pooled ribozymes
and which have an altered phenotype or response, and that only a
limited number of ribozyme species are recovered as compared to the
number of species present in the original library of pooled
ribozymes. Jurkat cells derived from the experiment described in
Example 11 which had been sorted for the lack of specific protein
induction were lysed in lx Taq PCR buffer/0.45% NP-40/0.45%
Tween-20, and ribozyme sequences were amplified from cellular DNA
in a standard PCR reaction using 1.times. Taq PCR buffer/1.5 mM
MgCl.sub.2/200 .mu.M dNTPs/0.2 .mu.M oligonucleotide primers/0.625U
Taq polymerase (Promega, Madion, Wis.). Amplification was performed
for 30 cycles, using a melting temperature of 94.degree. C. for 30
seconds per cycle, an annealing temperature of 60.degree. C. for 30
seconds per cycle, and an extension temperature of 72.degree. C.
for 45 seconds per cycle. A final incubation of 72.degree. for
seven minutes followed by the final amplification cycle. The primer
pair used to amplify the ribozyme sequences was derived from the
AAV plasmid vector sequences flanking the ribozyme insert, and had
the sequence 5'-ATCCGCGTCCTAGGCACGTGA-3' (SEQ ID NO: 44) and
5'-GTTACTAGTCCGCGGCTCGAC-- 3' (SEQ ID NO: 45). PCR products
containing ribozyme sequences were cloned into pCR2.1-TOPO
(Invitrogen, Carlsbad, Calif.). Cloned DNAs were transformed into
bacteria and the transformed bacteria were plated on LB-agar/amp
plates. Colony purified ribozyme clones were then sequenced, and
the identities of the ribozymes associated with the loss of cell
responsiveness were ascertained. While the original library of
pooled ribozyme sequences consisted of 12 or more individual
ribozyme specifies, the majority of clones (42/45) sequenced
contained ribozymes of a single species. Only one other ribozyme
species was represented in this particular analysis (3/45 clones).
These results indicate that isolation of specific ribozyme
sequences associated with the loss of a specific cell phenotype or
response is possible, even when these ribozyme sequences constitute
a minor component of a larger library of pooled ribozymes.
Knowledge of the specific ribozyme sequences associated with the
loss of cellular function can be used to clone and/or identify
previously known or unknown cellular genes involved in generating a
specific cellular phenotype or response using standard molecular
biologic techniques.
[0079] The above examples describe methods and compositions for
construction of a combinatorial ribozyme library and its high
throughput delivery and intracellular expression to determine the
function of a product(s) encoded by a target nucleic acid that
contains a motif of interest. Methods are described for design of
oligonucleotides that encode a combinatorial ribozyme library to
nucleic acids encoding proteins containing a motif of interest, the
construction of vectors that express nucleic acids that encode a
combinatorial ribozyme library; the ligation of the
oligonucleotides into retrovirus vectors, other viruses, or plasmid
vector; the packaging of the recombinant vector into virus
particles; the expression of the encoded library from cells either
infected with the virus particles or cells directly transfected
without a bacterial amplification step with the recombinant plasmid
expression vectors. The results demonstrate that a combinatorial
ribozyme library expressed from either recombinant virus or
recombinant plasmid expression vectors inactivate a target nucleic
acid to produce an altered cellular phenotype, and that both the
specific ribozyme species and the targeted cellular gene associated
with that altered cellular phenotype can be identified, so that a
function can be assigned to the target nucleic acid.
[0080] All publications and patent applications mentioned in this
specification are indicative of the level of skill of those skilled
in the art to which this invention pertains. All publications and
patent applications are herein incorporated by reference to the
same extent as if each individual publication or patent application
was specifically and individually indicated to be incorporated by
reference.
[0081] The invention now having been fully described, it will be
apparent to one of ordinary skill in the art that many changes and
modifications can be made thereto without departing from the spirit
or scope of the appended claims.
Sequence CWU 1
1
* * * * *
References