U.S. patent application number 12/234540 was filed with the patent office on 2009-03-26 for high-throughput methods for identifying gene function using lentiviral vectors.
This patent application is currently assigned to VIRXSYS CORPORATION. Invention is credited to Boro DROPULIC.
Application Number | 20090081682 12/234540 |
Document ID | / |
Family ID | 23005310 |
Filed Date | 2009-03-26 |
United States Patent
Application |
20090081682 |
Kind Code |
A1 |
DROPULIC; Boro |
March 26, 2009 |
HIGH-THROUGHPUT METHODS FOR IDENTIFYING GENE FUNCTION USING
LENTIVIRAL VECTORS
Abstract
The present invention relates to methods and compositions for
the efficient identification of one or more functionalities of a
product encoded by a nucleic acid sequence of interest. The methods
utilize the abilities to over and/or under express the product in a
cell, as well as the combination of these results, to permit the
identification of at least one of the product's cellular or in vivo
functionality.
Inventors: |
DROPULIC; Boro; (Ellicott
City, MD) |
Correspondence
Address: |
VIRxSYS;c/o MOFO SD
12531 HIGH BLUFF DRIVE, SUITE 100
SAN DIEGO
CA
92130-2040
US
|
Assignee: |
VIRXSYS CORPORATION
Gaithersburg
MD
|
Family ID: |
23005310 |
Appl. No.: |
12/234540 |
Filed: |
September 19, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10627940 |
Jul 25, 2003 |
7427474 |
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12234540 |
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PCT/US02/02287 |
Jan 25, 2002 |
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10627940 |
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60264272 |
Jan 25, 2001 |
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Current U.S.
Class: |
435/6.13 |
Current CPC
Class: |
C12N 15/1034
20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of identifying a function of a gene sequence of
interest in a cell type comprising (a) over expressing all or part
of said sequence in a first population of said cell type; (b)
inhibiting expression of said sequence in a second population of
said cell type; (c) detecting changes in one or more cellular
factors in said first and second populations; (d) identifying a
function of said gene sequence of interest based on the identity
of, or effect on, said one or more cellular factors.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
10/627,940, filed on Jul. 25, 2003 which is a continuation-in-part
("CIP") of PCT/US02/02287, filed Jan. 25, 2002 and designating the
United States, which claims benefit of priority to U.S. Provisional
Patent Application 60/264,272, filed Jan. 25, 2001, both of which
are hereby incorporated by reference in their entireties as if
fully set forth.
TECHNICAL FIELD
[0002] The present invention is directed to methods, as well as
compositions related thereto, for the efficient identification of
one or more functionalities of a product encoded by a nucleic acid
sequence. The methods utilize the abilities to over and/or under
express the product in a cell, as well as the combination of these
results, to permit the identification of at least one of the
product's cellular or in vivo functionality.
BACKGROUND ART
[0003] The tremendous efforts at sequencing the genomes of human
beings and other organisms has produced a vast amount of nucleic
acid and protein sequence information for additional analysis. Much
of the sequence information is now, or will be, the subject of both
biochemical and functional characterization. The sequence
information also serves as the raw material for "bioinformatics",
where the sequence itself is used in comparisons with other
sequences for which the structure, function, or other
characteristics have been previously identified. The great hope and
expectation for these efforts is that with the identification of
functionalities encoded by genetic sequences, additional
therapeutic products and treatments can be developed for diseases
in humans and other organisms.
[0004] The effort to identify functions encoded by genetic
sequences has focused, at least initially, on sequences that encode
actual gene products, or "genes". Earlier approaches sought to
clone and sequence only genes based on tools and strategies for
using positional cloning to map and clone genes. While labor
intensive, positional cloning has been successful in locating genes
associated with various diseases. Initially, genetic mapping is
performed based on large families of related individuals to locate
a disease associate gene at the level of chromosomal location and
in the range of centimorgans. Next, and with a significant increase
in effort, the work becomes one of physically mapping the genes so
that centimorgans are reduced to megabasepairs and then finally to
particular nucleotides. Examples of successes with positional
cloning include the identification of genes associated with cystic
fibrosis and Huntington's disease.
[0005] Other approaches to the isolation of genes include exon
trapping (Buckler et al. (1991) P.N.A.S. 88:4005-4009) and direct
selection (Morgan et al. (1992) N.A.R. 20:5173-5179). These methods
identify potential genes in large genomic regions which are then
sequenced and used in confirming the genes as actually expressed.
In some cases, cells that normally express the potential gene are
unknown, and it remains necessary to confirm the expression of the
genes and identify the functionality of the encoded product.
[0006] An initial advantage available with positional cloning over
the above two methods is that there is no need for knowledge
concerning the functional or physiological role of the gene product
of the identified gene. The identification is made based on
following a phenotypic trait followed by studying genetic
segregation of a particular sequence with the trait. But after
identification, there may still be difficulties in determining the
functional role of the gene product for the design of appropriate
therapies. Without knowing the functional role of the encoded
product, it remains difficult, for example, to identify suitable
agents to use as pharmaceuticals to appropriately target the gene
product. Additionally, it remains unknown how the identified gene
is involved in the progression from onset and progression to the
later stages of the disease.
[0007] A more recent approach to the isolation of genes has been
based on massive sequencing efforts designed to identify all
expressed sequences in a genome. Completion of such efforts in the
human and Drosophila genomes, as well as some microorganisms, have
been recently reported. But with the production of such large
amounts of sequence information, the need for a rapid and efficient
means for identifying the functionality of encoded gene products
increases further. This need has led to intensive commercial and
industrial activity for additional methods to identify gene
function.
[0008] One means for identifying function is through
bioinformatics, which seeks to determine functionality based on
similarities between a new sequence and other sequences for which
the structure, function, or other characteristics have been
previously identified. Bioinformatics is most often performed with
computer programs and thus have been termed to occur "in silico".
One drawback of bioinformatics, however, is that it only provides a
starting point for possibly validating a postulated functionality
of a gene sequence. Until a new sequence is actually expressed and
characterized within a living cell or organism, the supposed
functionality remains a hypothesis to be proven.
[0009] An approach to validate an assigned gene function is via the
use of small animal models. For example, transgenic mice have been
used for the overexpression of gene sequences in attempts to
identify the encoded functionality. Gene sequences have also been
used in the production of "knockout" mice where the endogenous
mouse sequence is no longer expressed. But the time and cost of
transgenic approaches have limited their usefulness to studies of
only a few sequences at a time.
[0010] Another approach has been to make use of cell cultures to
overexpress a gene sequence of interest. Unfortunately, there is no
rapid and efficient means for reliably producing a "knockout" cell
where the endogenous cellular sequence is not expressed or
overexpressed. Overexpression methods are, however, limited by the
vector system used to deliver and express the gene. As an initial
matter, known vector systems limit the number of cells that are
transfected with the gene. For example, plasmid vectors have low
transfection efficiencies and thus require the use of a selectable
marker to isolate transfected cells. But the expression of a marker
gene from the plasmid vector tends to skew the phenotype detected
because the gene of interest is not the only gene being
overexpressed in the cell. Stated differently, expression of the
gene of interest is not the only initial perturbation occurring in
the cell. As such, the determination of gene function may be
significantly mistaken due to skewing by expression of the marker
gene. The same selectable marker mediated skewing is seen with some
viral vectors, such as onco-retroviral vectors.
[0011] Higher transfection efficiencies are available from other
viral vectors, such as adenovirus based vectors, but these vectors
often fail to provide stable expression of the gene of interest.
More importantly, such vectors often have large numbers of their
own genes to express or suffer the risk of contamination due to
co-infection by helper virus. The expression of vector and/or
helper virus genes again perturbs the intracellular environment and
skews the detected phenotype and thus affects the determination of
gene function.
[0012] An additional limitation on the use of vector based
overexpression is found with the uncertainty as to what resultant
phenotype should be, or can be, detected in the transfected cell.
Moreover, such methods rarely use primary cells but instead use
cell lines or diseased cells where any identified gene function
remains suspect because of the abnormal cellular environment.
[0013] Citation of the above documents is not intended as an
admission that any of the foregoing is pertinent prior art. All
statements as to the date or representation as to the contents of
these documents is based on the information available to the
applicant and does not constitute any admission as to the
correctness of the dates or contents of these documents.
SUMMARY OF THE INVENTION
[0014] The present invention provides compositions and methods to
increase the ability to identify one or more functions of products
encoded by unidentified gene sequences or to further identify or
confirm one or more functions of known gene sequences. Therefore,
and in one aspect, the invention provides a lentiviral vector
capable of high transduction in primary cells, preferably without
altering the overall gene expression profile of the cell, except
for the expression of a specific payload encoding, or targeted to,
one or more gene sequences under investigation. Gene expression
profile refers to the levels of expression, at the RNA and/or
protein levels, of coding sequences in a cell.
[0015] The present invention thus provides a clear validating
system for the determination of gene function, where the cellular
effects of overexpression may be compared to and correlated with
those of inhibition. The invention may be applied, as a
non-limiting, but important example of a large scale gene chip
experiment where the background level(s) of gene expression is a
significant difficulty to data interpretation. The (cellular or
endogenous) genes identified or confirmed to be affected by
increased and/or decreased expression of a gene sequence of
interest can be placed in a matrix for analysis to describe the
function of the gene under investigation.
[0016] The present invention provides for the determination of one
or more functionalities of a given unidentified or known gene
sequence of interest by at least two means. First, the gene
sequence, or one or more portions thereof, is inserted in a vector
and introduced into a cell for expression of the encoded gene
product. The level of expression can of course be attenuated, but
preferably, the sequence is overexpressed. After expression occurs,
changes in the expression, composition, or form of endogenous
cellular factors, in comparison to normal cells without said
vector, are detected and analyzed. This permits the identification
of what cellular factors are affected by the sequence being
expressed or overexpressed. Without limiting the scope of the
invention, the actual effect on the cellular factor may include
that of changes in its level of expression (e.g. at the protein
and/or RNA levels), changes in its amino acid composition (e.g.
number and type of subunits and/or splice variants), and changes in
its state of post-translational modification (e.g. phosphorylation
and/or glycosylation and/or lipid modification) or location (e.g.
subcellular location as well as being soluble, membrane associated,
or by insertion of at least one portion of the factor into the
hydrophobic portion of a membrane). Cellular factors include those
with one or more identified function as well as those for which a
function has yet to be identified.
[0017] Second, expression of the unidentified or known gene
sequence is inhibited or terminated in a cell. Without limiting the
scope of the invention, the inhibition may be by use of all or part
of the gene sequence to recombine with the endogenous copy or
copies of the sequence in said cell to terminate its expression.
Alternatively, the gene sequence, or one or more portions thereof,
maybe inserted in an antisense orientation in a vector. The
expression of the sequence, or portion thereof, may be regulated
such that it is expressed only when desired to produce an antisense
nucleic acid.
[0018] Preferably, the antisense sequence is ligated to
co-localization sequences capable, upon expression with the
antisense sequence, of co-localizing the antisense sequence with
the complementary endogenous cellular, and "sense", sequence. In
some embodiments of the invention, the antisense sequence is used
to target a ribozyme to cleave the endogenous mRNA. The vector is
introduced into a cell for expression of the antisense sequence,
which then binds to and results in the inhibition of expression of
the complementary endogenous cellular sequence.
[0019] Alternatively, polynucleotides corresponding or
complementary to all or part of a gene sequence of interest may be
used in the design or testing or use of polynucleotides for
post-transcriptional gene silencing (PTGS). PTGS is mediated by the
presence of a homologous double stranded RNA (dsRNA) which leads to
the rapid degradation of RNAs encoding a targeted gene product. One
form of PTGS is RNA interference (RNAi) mediated by the directed
introduction of dsRNA. Another form is via the use of small
interfering RNAs (siRNAs) of less than about 30 nucleotides in
double or single stranded form that induce PTGS in cells. A single
stranded siRNA is believed to be part of an RNA-induced silencing
complex (RISC) to guide the complex to a homologous mRNA target for
cleavage and degradation. siRNAs induce a pathway of gene-specific
degradation of target mRNA transcripts. siRNAs may be expressed in
via the use of a dual expression cassette encoding complementary
strands of RNA, or as a hairpin molecule.
[0020] Therefore, the invention also provides for methods of
inhibition or termination of expression of a gene sequence by the
use of short interfering (si) RNAs or ribozymes targeted against
said sequences. The use of ribozymes to inhibit gene expression and
virus replication is described in U.S. Pat. No. 6,410,257 via use
of a conditionally replicating vector for other purposes.
[0021] After expression of the antisense, ribozyme, or siRNA
sequence(s) to inhibit expression of the complementary cellular
sequence, changes in the expression, composition, or form of
cellular factors as described above, in comparison to untreated
normal cells, are detected and analyzed. This permits the
identification of what cellular factors are affected by decreasing
or suppressing expression of the endogenous cellular sequence
corresponding to the gene of interest (complementary to the
antisense sequence used).
[0022] Preferably, the above over and underexpression of a gene
sequence of interest is conducted by use of a viral vector capable
of high efficiency transduction without significant expression of
endogenous vector gene sequences or helper virus contamination.
Examples of such vectors include those described in pending U.S.
patent application Ser. No. 09/667,893 entitled "Improved
Conditionally Replicating Vectors, Methods for Their Production and
Use", filed Sep. 22, 2000, which is hereby incorporated by
reference as if fully set forth. Even more preferred are
embodiments of the invention wherein the transduced cells are
primary cells.
[0023] Optionally, the above vectors for over and underexpression
are integrated into the cellular genome as part of the transduction
process.
[0024] Alternatively, the vectors of the invention, such as a
lentiviral vector, may be used to introduce more than 1) a single
inhibitory or terminating sequence, 2) an overexpressed gene
sequence, or 3) a combination of the two. Nucleic acid constructs
for the expression of such multiple sequences may contain a
separation of the gene sequences by transcriptional pause elements,
stop elements, by a (native) cis-acting ribozyme that self cleaves
the transcript between the two encoded RNAs, or by a combination of
these elements. Alternatively, dual vectors may be used to target
the same cell in order to allow simultaneous gene knockdown,
expression, or a combination of knockdown and expression.
[0025] In a preferred form of the invention, changes in the
expression of cellular factors are detected. Additionally, the
detected changes in expression of cellular factors from the two
approaches can be combined and compared to provide additional
information on one or more functions of the unidentified or known
gene sequence under study. The combination of the detected changes
in expression of cellular factors is similar to "subtraction"
techniques used to study the differential expression of cellular
factors upon a perturbation in cellular conditions, such as before
or after a temperature shift or the addition of a growth
factor.
[0026] Detailed analysis of the results from overexpressing,
underexpressing and the results from both permits the
identification of one or more gene functions of a sequence of
interest based on a reliable intracellular environment initially
perturbed only by changes due to over or under expressing the gene
sequence of interest. A function of said gene sequence of interest
is thus identified based on the identity of, or effects on, one or
more cellular factors affected by changes in the expression of said
sequence. Non-limiting examples of possible functions include
regulating the expression of said one or more factors and affecting
the activities of said one or more factors.
[0027] The analysis also permits the identification of one or more
cellular factors that are functionally related to the sequence of
interest. One such group of cellular factors would exhibit
increased expression upon over expression of the sequence of
interest and exhibit decreased expression upon inhibition of
expression of the sequence of interest. Another group of cellular
factors would be the inverse of the above, exhibiting decreased
expression upon over expression of the sequence of interest and
exhibiting increased expression upon inhibition of expression of
the sequence of interest.
[0028] The groups of cellular factors that are thus identified may
be viewed as part of a "coordinated response" to perturbations in
the expression of the sequence of interest. The "coordinated
response" may be that of a single regulatory, biochemical or
metabolic pathway or other functionality of a cell. It also
provides a means for the identification of functional relationships
between cellular factors and the product of the gene sequence of
interest.
[0029] The ability to identify "coordinated response" cellular
factors by observing the effects of both over and underexpression
of a sequence of interest provides an advantageous means of
decreasing or eliminating time spent on evaluating or considering
cellular factors that display a change in expression only upon
either the over expression, or under expression, of a sequence of
interest. Such "coordinated response" cellular factors may be
readily classified as a separate group for separate study,
consideration, and/or analysis. The present invention improves the
ability to quickly and efficiently identify functionalities of the
gene sequence of interest since it decreases the expense in time
and money spent on simultaneously relating all the effects of
perturbing the expression of the sequence of interest. The
invention provides a means to focus only on those effects that are
correlated with both the over and under expression of the gene
sequence of interest.
[0030] The invention may be practiced by detecting changes in one
or more cellular factors of a cell or cell type in which the gene
sequence of interest has already been found to be expressed. A
non-limiting example of such a gene sequence of interest is in the
case of an open reading frame which is found to be expressed in
certain cell types or under certain disease conditions.
Alternatively, the invention may be practiced by detecting changes
in a cell or cell type in which the gene sequence of interest has
not been detected as expressed. Preferably, the cells or cell types
are human cells, although any animal, plant or microorganism cell
may also be used. Methods for the introduction of a gene sequence
of interest into a cell are discussed below.
[0031] The present invention thus provides analytical methods,
compositions and systems comprising two or more vectors for the
identification of one or more functionalities of a gene sequence of
interest. Optionally, at least a third vector is used to over or
under express yet another gene sequence to provide further
information on one or more functionalities of a gene sequence of
interest.
[0032] In another aspect of the invention, a high throughput, and
optionally computerized or robot implemented, system for
identifying gene function is provided. In such embodiments, the
invention provides libraries of vectors and transduced cells
arranged in a multiplicity of compartments. With respect to
vectors, the libraries contain compartments containing either a
vector for overexpressing a gene of interest or a vector for
underexpressing a gene of interest. Such vector libraries may be
very efficiently used to transduce cells to produce a library of
cells in a multiplicity of compartments, each of which contains
cells transduced with one vector. The vector libraries may
optionally be propagated in packaging cells prior to their use in
cell transduction.
[0033] The libraries of transduced cells may be analyzed for the
effects of over or under expressing a gene sequence of interest by
use of machine implemented microarray or macroarray technologies
known in the art. An example of which is "gene chip" technology
whereby gene expression of a large number of sequences may be
determined via a single "chip" used for the hybridization of mRNA,
or the corresponding cDNA, isolated from cells. The invention
includes a composition of matter that is an array for the practice
of the disclosed methods, optionally in contact with material from
cells that are over and/or under expressing one or more gene
sequence of interest (e.g. in contact with RNA, protein, other
cellular material, or extracellular material from such cells).
[0034] The libraries of transduced cells may also be subject to
further treatment or changing conditions before analysis of effects
on cellular factors. The cells, and hence effects on cellular
factors, may also be analyzed temporally. The function of a gene
sequence may also be assessed through cellular differentiation and
function in vivo in culture, or after transplantation in an animal
model, or in human or non-human primates.
[0035] A variety of methods may be used to detect changes in
cellular factors. Such methods include the determination of
messenger RNA levels, protein expression levels, protein activity
levels, effects on protein phosphorylation, effects on protein or
nucleic acid processing, effects on RNA stability, effects on
signal transduction or second messengers, and so forth.
[0036] The invention also provides methods for altering the
expression, composition, or form of one or more cellular factors in
a cell by over expressing, inhibiting the expression of, or
simultaneously inhibiting and overexpressing a gene sequence or
sequences for which a function has been identified by the methods
described above. Such methods may also be used to alter the
phenotype of said cell.
[0037] The invention provides numerous advantages beyond the
ability to identify one or more functions of encoded gene products
for which no activity is known. These include the ability to
provide additional information on the function of gene products for
which some activity information is already known; the ability to
provide information on the effect of over or under expressing one
functionless gene product on the expression of another functionless
gene product; and the ability to conduct the same analysis on
different cell types which express different endogenous
sequences.
[0038] The invention also provides a means for increasing the
expression of known gene products. Once a gene sequence of interest
has been found to increase expression of a desirable and known
cellular gene product, the gene sequence of interest may be used at
least to increase expression of the product for subsequent
isolation or purification.
[0039] It is a further advantage of the present invention that
there is no requirement for knowledge or speculation on the
functionality of the gene of interest. In embodiments of the
invention where there is knowledge concerning the functionality of
the gene of interest, the present invention advantageously provides
means to identify one or more other functionalities that may have
been previously unknown and/or to confirm one or more other
functionalities that may have been previously known or suspected.
The latter is of particular relevance with respect to a disease
associated gene sequence of interest which can be used in
combination with the present invention to identify or confirm one
or more other functionalities of the sequence. For example, and
without limiting the invention, a decrease in the level of a
product encoded by a disease associated gene sequence may have been
identified as a useful pharmacological treatment for the disease.
But a decrease in the expression level of the sequence may be
suspected of causing a compensatory increase in another cellular
factor which would decrease the efficacy of the treatment. Use of
the disease associated gene sequence in the present invention
provides an advantageous means of determining whether such a
compensatory increase occurs as well as the identity of the
compensatory cellular factor. This factor is a second target which
may be simultaneously decreased to improve the treatment of the
disease.
[0040] Yet another advantage of the invention is that relatedness
based on gene functionality may be determined and used to produce a
map of functional relationships.
BRIEF DESCRIPTION OF THE FIGURES
[0041] FIG. 1 shows sample results when various sequences of
interest, "Seq" 1 to 4, are over or under expressed. The effects on
the expression of various cellular gene sequences are depicted
along with the level of expression in control cells indicated as
"100" in arbitrary relative units. In this FIGURE, "Seq" 1-4 may
represent sequences that are unidentified, putatively identified
and/or known. The results may be increased at will based upon
inclusion of more cellular gene sequences for evaluation (more rows
added) or more sequences of interest to over and under express
(more columns added).
MODES OF CARRYING OUT THE INVENTION
[0042] The present invention provides methods and compositions for
the identification of one or more functionalities of the gene
product of a given sequence. Preferably, the sequence is human, but
one or more non-human sequences may also be used in combination
with the present invention to identify their effect(s) on cellular
factors in human cells. Advantageously, there is no prerequisite
for knowledge regarding the encoded functionality. If the
functionality is known, however, the present invention permits the
confirmation of said functionality as well as the possible
identification of previously unknown or unappreciated
functionalities.
[0043] In a preferred embodiment, the invention provides a vector
for overexpressing a given unidentified or known gene sequence in a
cell. Such expression is preferably under tight and/or inducible
regulatory control. An "unidentified" sequence is considered to not
yet have confirmation of a cellular or biochemical functionality. A
"known" sequence is considered to have been confirmed as having one
or more cellular or biochemical functionalities. Preferably, the
overexpression occurs without simultaneous expression of other
vector borne sequences, such as, but not limited to, selectable
markers. Thus the intracellular environment is affected only by the
overexpression of the sequence of interest and the effects of said
overexpression more accurately reflect one or more functionalities
of said sequence.
[0044] Cells transduced according to this embodiment of the
invention are analyzed for cellular factors, defined herein as any
cellular gene product (e.g. proteins or RNA) or metabolite thereof
(e.g. molecules such as sugars and lipids), that are affected by
overexpression of said gene sequence. The effects of overexpression
are in comparison to normal cells not overexpressing said sequence.
Preferably, normal cells are mock transfected with the vector but
without expression of said gene sequence. By way of example, and
without limiting the invention, overexpression of a given gene
sequence (such as that encoding an inducer of cellular
differentiation) would increase expression of RNAs encoding one or
more cellular factors (such as those encoded by genes involved in
differentiation or the differentiated state) in comparison to
normal cells. Alternatively, overexpression of some gene sequences
(such as a transcriptional repressor) would result in decreased
expression of one or more cellular factors. Lastly, some cellular
factors are unaffected by overexpression of some gene sequences.
The invention includes the ability to identify one or more
functions of gene sequences of interest that encode modulators of
one or more cellular factors by binding to nucleic acids encoding,
or regulating the expression of, said factor(s).
[0045] In another embodiment, the invention provides a vector for
inhibiting, suppressing or otherwise decreasing the expression of
an unidentified or known gene sequence in a cell. This again
preferably occurs in the absence of expression of other vector
borne sequences, such as, but not limited to, selectable markers.
The intracellular environment is thus again only affected by the
complete or partial underexpression of said sequence, and the
effects more accurately reflect one or more functionalities of said
sequence. While this underexpression of a gene sequence appears to
require that the cells normally express the sequence endogenously,
the present invention may still be practiced with cells that do not
express the sequence because there would simply be no significant
difference between the cells transduced with vector to effect
underexpression and mock transduced cells. Alternatively, cells
that normally express the sequence endogenously, and thus are
capable of underexpressing it, may be first identified by well
known and standard methods in the art such as a Northern blot using
all or part of the sequence as a probe. To identify such cells
rapidly, a "tissue blot", wherein RNA from a variety of cell types
is prepared and simultaneously subjected to Northern blotting, may
be used.
[0046] To underexpress the unidentified or known gene sequence, but
without limiting the invention, it may be inserted in an antisense
orientation in a vector for transduction and expression in a cell.
Such expression is preferably under tight and/or inducible
regulatory control. The insertion of the entire sequence in
antisense orientation is of course not necessary and one or more
portions of the unidentified or known sequence may be used.
Preferably, the antisense sequence is operably linked to
co-localization sequences which, upon expression with the antisense
sequence, of co-localizing the antisense sequence to be tracked to
the same cellular locations as the complementary endogenous
cellular, or "sense", sequence. While the antisense sequence can be
used directly to result in the non-expression of the endogenous
mRNA, the antisense sequence can also be part of the targeting
sequence to direct a ribozyme to cleave the endogenous RNA. In such
embodiments, the vector is of course designed to be able of
expressing the antisense sequence as an operative part of an
encoded ribozyme to target the endogenous sequence. The vector is
then introduced into a cell for expression of the antisense
sequence, which then binds to and results in the inhibition of
expression of the complementary endogenous cellular sequence.
[0047] A variety of antisense sequences derived from various
portions of the gene sequence to be suppressed may be used
initially to determine which is most suitable for decreasing the
expression of a cellular sequence. In one embodiment of the
invention, and for the most complete suppression of endogenous
cellular expression, the antisense sequence should be directed to a
conserved portion of the endogenously expressed sequence in case
the cell is heterozygous for the gene sequence being suppressed. Of
course multiple antisense sequences may also be used.
Alternatively, the gene sequence of interest may be used to prepare
vectors that would recombine with the endogenous copies of the gene
sequence of interest to suppress their expression.
[0048] While a variety of co-localization sequences may be used to
co-localize the antisense molecule to the endogenous RNA, preferred
sequences are the U1, U2, U3, U4, U5 or U6 snRNA, all of which may
be operably linked to the above described antisense or ribozyme
sequences. More preferably, the co-localization sequence used is a
U1 snRNA/promoter cassette as described in Dietz (U.S. Pat. No.
5,814,500), which is hereby incorporated by reference in its
entirety as if fully set forth.
[0049] While for many gene sequences, the ability to suppress its
expression entirely provides the clearest information on the
results of its underexpression, it should be noted that the ability
to suppress, partially or entirely, the expression of a sequence is
an aspect of the present invention. Partial suppression of gene
expression is of particular advantage when the gene sequence
encodes a product critical for cell viability. Such gene sequences
may be readily identified by the lethal effect on a cell upon
complete or nearly complete suppression of expression. A
non-limiting example of how to achieve partial suppression is to
target only one endogenously expressed sequence in a cell that is
heterozygous for said sequence.
[0050] Cells transduced according to this embodiment of the
invention are analyzed for cellular factors that are affected by
underexpression of said gene sequence in comparison to normal cells
expressing said sequence. The normal cells are again preferably
mock transfected with the vector but without causing
underexpression of said gene sequence. By way of example, and
without limiting the invention, underexpression of a given gene
sequence (such as that encoding a transcriptional repressor) would
increase expression of RNAs encoding one or more cellular factors
(such as those encoded by genes repressed by said repressor) in
comparison to normal cells. Alternatively, underexpression of some
gene sequences (such as transcriptional activators) would result in
decreased expression of one or more cellular factors. Lastly, some
cellular factors are unaffected by underexpression of some gene
sequences.
[0051] While not absolutely necessary for the practice of the
invention, vectors for over or under expressing sequences in accord
with the present invention are preferably capable of high
efficiency and stable transduction of cells of up to 100%
efficiency. Alternatively, they are maintained episomally,
preferably at high copy number although the invention may also be
practiced with low copy number episomal constructs. Stable
integration may be enhanced by stimulating the cells being
transduced with an appropriate ligand followed by culturing the
cells under standard conditions (see co-pending U.S. application
Ser. No. 09/653,088 filed Aug. 31, 2000 and titled METHODS FOR
STABLE TRANSDUCTION OF CELLS WITH VIRAL VECTORS, and allowed in
June 2003) which is hereby incorporated in its entirety as if fully
set forth. Such vectors are also preferably designed to express
little or no vector borne sequences other than the gene of
interest, whether in sense or antisense orientation. In some
embodiments of the invention, the vectors further contain sequences
sufficient to permit integration of the vector into the cellular
genome. Such recombination events may be based on homologous
recombination or integrase mediated events due to enabling
sequences present on the vector. As a non-limiting example, when a
Lentiviral derived vector is used, the normal Lentiviral
integration sequences can facilitate stable integration into the
host cell genome. Such Lentiviral vectors are preferably
pseudotyped by use of a heterologous viral envelope (env) protein,
such as, but not limited to, that of a retrovirus. More preferably
the env protein is an HIV-1, HIV-2, or MMLV envelope protein; the G
protein from Vesicular Stomatitis Virus (VSV), Mokola virus, or
rabies virus; GaLV, Alphavirus E1/2 glycoprotein, or RD114, an env
protein from feline endogenous virus. Alternatively, sequences
encoding a chimeric envelope protein may also be used. Sequences
encoding an envelope protein from the following viral families may
also be used: Piconaviridae, Tongaviridae, Coronaviridae,
Rhabdoviridae, paramyxoviridar, Orthomixoviridae, Bunyaviridae,
Arenaviridae, Paroviridae, Poxviridae, hepadnaviridae, and herpes
viruses.
[0052] The given unidentified or known gene sequence to be over or
under expressed can be from any source and may even be partially
identified. Non-limiting examples of unidentified or partially
identified sequences include those obtained from the isolation and
characterization of EST (expressed sequence tag) sequences and any
nucleic acid sequence considered to possibly encode a gene product,
whether RNA or proteinaceous in form. Such sequences include those
identified by the assembly of EST sequences or otherwise determined
to encode a gene product. These sequences include those that have
undergone bioinformatics analysis and thus have homology to other
known or uncharacterized sequences. By way of example, and without
limiting the invention, a sequence encoding an open reading frame
for which no function is assignable may be used in the present
invention to identify one or more of its functions in a cell.
Similarly, a sequence encoding an open reading frame with homology
to a DNA binding protein (based on bioinformatics analysis, for
example) may be used in the present invention to confirm its
putative functionality as a transcription factor.
[0053] Non-limiting examples of known sequences may be from any
source and include those for which one or more functionalities have
been assigned. Such sequences include those in publicly available
databases as well as any sequence for which the encoded gene
product has been characterized. Such sequences may nevertheless be
used in the present invention to confirm known functionalities
and/or identify additional functionalities. By way of example, and
without limiting the invention, a sequence encoding a kinase
identified solely as phosphorylating a cytoplasmic protein may be
found to cause elevated expression of a nuclear transcription
factor upon overexpression of the kinase. Without being bound by
theory, the kinase may directly or directly result in the increased
expression of a transcription factor via its kinase activity. One
possibility would be where the kinase phosphorylates the
transcription factor to inactivate it, thereby causing an increase
in its expression via a feedback loop. Other effects on cellular
factors as described herein may also occur via one or more feedback
loops.
[0054] Additionally, artificial sequences, such as recombinant
fusion or other chimeric constructs as well as mutated versions of
the sequences discussed above, may also be used in the present
invention to identify their function(s). This aspect of the
invention may be of particular advantage in the confirmation of a
particular artificial protein or mutagenized protein as capable of
substituting for the function(s) of a wildtype protein. For
example, and without limiting the invention, a synthetic mutant
version of the p53 protein which is able to multimerize with itself
but not with dominant negative mutant forms of p53 may be used in
the present invention to confirm its ability to substitute for
wildtype functional p53. With such confirmation, the synthetic
mutant may be used in therapeutic contexts to treat cells
containing the dominant negative p53 mutation.
[0055] The introduction of unidentified or known sequences into the
vectors for the practice of the invention may be by any means.
Preferably, it is performed by highly efficient means that may be
performed in parallel and minimize the need for multiple cloning
steps or the need for confirmation of cloning steps. More
preferably, the insertion of sequences into vectors is performed by
automated techniques. As a non-limiting example, the gene sequence
of interest may be first cloned into an initial vector capable of
allowing the sequence to be subsequently introduced into the over
and under expression vectors of the invention. This may be by the
use of a recombination mediated insertion system such as the
Gateway.TM. cloning system from Life Technologies, which utilizes
att sites in the plasmids to permit highly efficient transfer of
sequences between vectors. Thus in one embodiment of the invention,
the vectors for over and under expressing a gene sequence may
contain appropriate att sites to permit efficient insertion of gene
sequences.
[0056] In an automated embodiment, the insertion of gene sequences
may be based upon the use of arrays containing a library of gene
sequences. Such sequence containing arrays may be used to generate
a plurality of additional arrays, organized based upon the first
library containing the gene sequences. This plurality of arrays may
sequentially include one or more of the following: an array that
contains the gene sequences modified with appropriate linkers; an
array that contains the modified gene sequences for amplification;
an array that contains the modified sequences introduced into an
initial vector for propagation or further cloning; an array of the
sequences transferred from the initial vector to one or more
vectors of the invention; and an array of such vectors
appropriately packaged prior to their use to transduce cells.
[0057] One advantage provided by the use of such arrays is the
ability to continue to use the organization present in such arrays
when over and under expressing a library of gene sequences
according to the invention. For example, the array arrangement
containing the library of packaged vectors can be used to transduce
an array of cells, which can then be harvested, partially or
completely, to analyze the effects of over and under expression of
the gene sequences of the array on cellular factors.
[0058] Cells for use in the present invention may be any kind of
cell. But for optimal determination of function, the cell should be
from the same organism as the gene sequence to be over or under
expressed. Sequences may nevertheless be heterologous to the cells
in which they are express to determine their function(s) in the
cell. Preferably, the cells are human, and the gene sequence of
interest is studied at least initially in cells from which the
sequence has been found to be expressed. By way of a non-limiting
example, a fungal sequence may be expressed in mammalian cells to
determine its function(s) therein. This aspect of the invention is
of particular advantage if the counterpart mammalian sequence to
the fungal sequence is known. This permits a comparison to the
effects of underexpressing the mammalian sequence to confirm the
fungal sequence as capable of functioning as a substitute for the
mammalian sequence. If so, the fungal sequence may encode a product
which may be a therapeutic substitute for the product encoded by
the mammalian sequence.
[0059] Preferred cell types for the practice of the invention are
eukaryotic cells, more preferred are primary eukaryotic cells, and
most preferred are primary mammalian cells and human cells.
Preferred cells are those of human tissues, including, but not
limited to, neuronal cells, brain cells, epithelial cells,
connective tissue cells (e.g. fibroblasts, osteoblasts, and adipose
cells), blood cells (e.g. leukocytes, lymphocytes, monocytes and
neutrophils), sensory cells, muscle cells, sensory cells (e.g.
ocular cells and hair cells), lung cells, heart cells, liver cells,
skin cells, pancreatic cells, breast cells, kidney cells,
intestinal cells, stomach cells, colon cells, prostate cells,
ovarian cells, and germ cells. Cultured cell lines, including those
derived from any of the above, may also be used. In another aspect
of the invention, however, partially and fully differentiated cells
may also be used if desired. By way of a non-limiting example, the
use of differentiated cells is preferred if the gene sequence to be
underexpressed is normally only expressed in said differentiated
cells. For the transduction of different cell types, the vectors
may be appropriately packaged via the use of pseudotype and
amphotropic packaging systems known in the art.
[0060] The ability to transduce a variety of cell types provides
another advantage of the present invention, wherein the over and
under expression of a gene sequence in a variety of (heterologous)
cell types may be used to provide added information and thus
enhance assignment of gene function. This enhancement is due in
part to the differences in endogenous gene expression in different
cell types. Thus the full range of functionalities for a gene
sequence may be better elucidated by evaluating its over and under
expression in a variety of cell types.
[0061] The expression of a gene sequence of interest in a
heterologous cell based upon one of more functions as identified by
the present invention also provides a means to alter the phenotype
of said cell. As a non-limiting example, over expression of a gene
sequence may result in the elevated expression of a cell surface
marker in cells normally expressing the sequence. In a heterologous
cell that normally does not express the sequence, expression of the
sequence therein may result in expression of the cell marker on the
surface of the heterologous cells, thus providing a novel way to
identify and/or target those heterologous cells.
[0062] In one preferred embodiment of the invention, the above
vectors for over and under expressing a gene sequence are
integrated into the cellular genome as part of the transduction
process.
[0063] In yet another aspect of the invention, the detected changes
in expression of cellular factors from over and under expression of
a sequence can be compared to provide additional information on the
functionality of the gene sequence under study. In FIG. 1, for
example, the overexpression (O) of unidentified sequence 2 ("Seq
2") is shown as increasing the expression of "structural protein
1". But the underexpression (U) of Seq 2 is shown as having a very
minor effect on "structural protein 1" expression compared to the
control cells (Con). As such, the relationship between Seq 2 and
structural protein 1 may be one where Seq 2 functions to activate
or otherwise induce expression of structural protein 1 while the
underexpression of Seq 2 has minimal effects on the background
expression of structural protein 1.
[0064] Similarly, the functional role of the product encoded by a
sequence may be analyzed by reviewing what cellular factors are
similarly affected. In FIG. 1, for example, over and under
expression of sequence 1 ("Seq 1") affects the expression of
"transcription repressor 1" and "transcription repressor 2"
identically. Thus the expressed product of Seq 1 functions to
regulate these two repressors in the same way. On the other hand,
the over and under expression of Seq 1 has an opposite effect on
"transcription factor 2" expression. This suggests that Seq 1
functions to simultaneously regulate cellular expression of the two
repressors and "transcription factor 2".
[0065] Moreover, the present invention provides a means of
identifying functional relationships between unidentified
sequences. In FIG. 1, for example, "Seq 3 and "Seq 4" have
identical effects on the expression of "oxidoreductase 1". This
would indicate that the expressed products of Seq 3 and Seq 4 are
functionally related to each other at least to the extent that both
function in the regulation of "oxidoreductase 1" expression.
Furthermore, overexpression of "Seq 2" in FIG. 1 is shown as
increasing the expression of "Seq 3" (see Seq 2's O, U and C
columns for row Seq 3).
[0066] The results in FIG. 1 also illustrate other functionalities
of a gene sequence. For example, "Seq 4" is shown as autoregulating
its own expression when its own level of expression is analyzed.
Overexpression of Seq 4 does not result in as much Seq 4 RNA
expression as compared to when Seq 1, 2 or 3 are overexpressed
(compare the four rows for Seq 1, 2, 3 and 4 against the identical
Seq columns). This would exemplify situations where the
overexpression of Seq 4 results in feedback inhibition of
endogenous Seq 4 expression. Similarly, underexpression of Seq 4
does not eliminate Seq 4 expression because of feedback activation
of endogenous Seq 4 expression.
[0067] The detected changes in expression of cellular factors can
also be combined to provide additional information on functional
relationships. As a non-limiting example, subtractive hybridization
can be used quantitatively to determine the difference in the
expressed RNAs between cells overexpressing and underexpressing a
gene sequence. For example, the total expressed RNA from a first
group of cells overexpressing a gene sequence can be used to
generate cDNA for subtractive hybridization against the total
expressed RNA from a second group of cells underexpressing the gene
sequence. If the amount of a particular RNA is higher in the cells
of the first group than the second group, there will be an excess
of cDNA corresponding to that particular RNA left as single
stranded molecules after hybridization. This cDNA can then be
isolated and detected. The subtractive hybridization is preferably
also performed using cells underexpressing a gene sequence as the
first group and cells overexpressing the sequence as the second
group. The results of such subtractive hybridization is shown in
FIG. 1, where (if applicable) there are two numbers for each
unidentified sequence "Seq" under the "C" column. The first number
refers to subtractive hybridization using cDNA from the
overexpressing group (O) and the second number refers to using cDNA
from the underexpressing group.
[0068] Additionally, subtractive hybridization can be also used to
compare the expressed RNAs between control cells and those either
over or under expressing a particular gene sequence. Thus RNAs
expressed in control cells can be "subtracted" from RNAs expressed
in cells over or under expressing a gene sequence to provide
additional information on the function of said gene sequence. This
approach may also be advantageous for the cloning of RNAs that are
differentially expressed between normal cells and those over or
under expressing a particular gene sequence.
[0069] The results in FIG. 1 can also be modified by placing the
cells under different culture conditions. By way of non-limiting
examples, the cells can be placed under active growth and/or
proliferation conditions, quiescent conditions, temperature shifted
conditions, and in the presence of a ligand conditions before the
RNA is prepared. The use of such conditions provides additional
information for determining one or more functionalities of a gene
sequence of interest.
[0070] In another aspect of the invention, one or more additional
gene sequences are simultaneously over or under expressed in
combination with the over or under expression of a first gene of
interest. As a non-limiting example, and based on FIG. 1, cells
transduced with a vector that overexpresses Seq 1 may instead be
separately transduced with vectors that simultaneously either over
or under expresses another sequence (e.g. "Seq 5"). Similarly,
cells transduced with a vector that underexpresses Seq 1 may
instead be separately transduced with vectors that simultaneously
either over or under expresses "Seq 5". Such simultaneous over or
under expression techniques provides additional information to
identify or confirm the function(s) as well as functional
relationship(s) of any gene sequence.
[0071] In another embodiment of this simultaneous approach, at
least a third vector may be used to simultaneously over or under
express the one or more additional gene sequences. Of course the
vector would be one that is compatible with the vector(s) used to
over or under express the first gene sequence. In yet another
embodiment of this simultaneous approach, the first gene sequence
may be closely related to the one or more additional gene sequences
being simultaneously over or underexpresses. As a non-limiting
example, the first gene sequence may be a wildtype sequence, the
cell used may be homozygous for a misfunctioning mutant of the
sequence, and the additional gene sequence to be expressed is an
antisense version of the endogenous sequence encoding the
misfunctioning mutant. By simultaneously expressing the wildtype
sequence and underexpressing the misfunctioning mutant sequence by
use of the additional gene sequence, the wildtype activity of the
first gene sequence may be restored to the cell.
[0072] In another embodiment of the simultaneous approach, the
additional gene sequence may encode an oncogene or a tumor
suppressor gene.
[0073] An another aspect of the invention is the use of a high
throughput system for the practice of the present invention. In one
embodiment of this aspect, the system maybe optionally computerized
or robot implemented, and may also include the use of the arrays
described above. In one embodiment of this approach, the invention
provides libraries of gene sequences, over and under expression
vectors containing them, cells transduced with said vectors, and
the effects on cellular factors by analysis of said cells.
Preferably, the libraries of gene sequences are present in a
multiplicity of compartments, each of which contains one gene
sequence. In a particularly preferred format, the compartments are
in a multi-well vessel, such as, but without limiting the
invention, a multi-well plate. Such a multi-well vessels may be
considered arrays containing all or part of gene sequence
libraries, and the organization of sequences present in such arrays
may be maintained throughout the practice of the invention, up to
and including the analysis on the effects on cellular factors. Of
particular advantage for the practice of the invention is the use
of vectors containing only one gene sequence to transduce cells in
each compartment.
[0074] In another aspect of the invention, separate arrays may be
used for over and under expressing a gene sequence of interest. But
the effects on cellular factors contained in such separate arrays
is preferably combined to provide greater ease of analysis. As a
non-limiting example, and once the effects of over and under
expression of a gene sequence are determined for each sequence of a
library, the information can be combined prior to further analysis
of the results. For example, FIG. 1 shows the combination of the
effects on a large number of cellular factors (see left column) of
over (see columns "O") and under (see columns "U") expression for
sequences 1-4 of a library (see top row). The actual effects on
cellular function can also be combined by means such as the
"subtractive hybridization" discussed above and then simultaneously
analyzed with the over and under expression data (see for example
columns "C" in FIG. 1).
[0075] In an additional approach for the practice of the invention,
the effects of over and under expression on cellular factors is
performed on micro or macro arrays capable of being machine
implemented. Such machines are preferably capable of being
partially or completely automated to harvest cells over or under
expressing a gene sequence to determine the effect(s) on cellular
factors. In a non-limiting example for analyzing the effect on gene
expression, a "gene chip" containing sequences encoding cellular
factors is used to determine which of these factors is affected by
over or under expressing a particular gene sequence. Thus RNAs, or
cDNAs corresponding thereto, may be isolated from the cells,
labeled, and hybridized against the sequences on said chip. The
results of such hybridization can be compared to that seen with
control cells to determine the effect on each cellular factor
encoding sequence present on the chip. Of course a multiplicity of
chips may be used to permit analysis of the large number of
cellular factors known, as well as permit the analysis of each
unidentified sequence against other unidentified sequences.
Additionally, duplicates of the same chip are used for analysis of
cells either over or under expressing a particular gene
sequence.
[0076] Prior to analysis, the libraries of transduced cells, which
over and under express a variety of sequences, may be subjected to
further treatment or changing conditions. In addition to the
simultaneous over or under expression of additional sequences
described herein, the cells may be subjected to the presence of
various factors and cultured under a variety of growth conditions.
As a non-limiting example, the cells may be exposed to one or more
ligands to induce a variety of effects. Alternatively, the cells
may be analyzed over time or transplanted into an in vivo context
to permit the identification of additional effects on cellular
factors.
[0077] In additional embodiments of the invention, the analysis of
effects on cellular factors may be conducted by the use of any
assay. The following is provided as additional non-limiting
examples of the practice of the invention. Of course these examples
may be conducted by partially or completely automated means.
[0078] In a first non-limiting example, cells over or under
expressing a gene sequence may be analyzed for the effects on
protein levels of cellular factors. As such, a sample of the cells
may be used in western blot analysis using antibodies specific for
various cellular factors. Alternatively, the analysis may be
conducted by other means, such as any quantitative immunoassay.
Such an analysis may be done in concert with the gene expression
analysis described herein to provide a more complete picture of
effects on cellular factors since changes in RNA expression levels
may not always be closely correlated with changes in the levels of
the protein encoded by said RNA. Moreover, this approach can follow
the gene expression analysis by using only antibodies directed to
proteins encoded by RNAs which have been observed to change in
expression.
[0079] In a second non-limiting example, cells over or under
expressing a gene sequence may be analyzed for the effects on
protein activity. This may be of particular interest for gene
sequences encoding an activator or inhibitor of another protein or
enzyme. A sample of the cells may be used in enzymatic or other
protein assays to detect changes in activity. For instance, the
over expression of an activator of a particular kinase would
increase the detectable activity of said kinase in an appropriate
assay. This effect may or may not be independent of any changes in
the gene expression or protein levels of the kinase.
[0080] In a third non-limiting example, the effect on protein
phosphorylation may be analyzed in cells over or under expressing a
gene sequence. The cells over or under expressing a gene sequence
may be grown such that phosphorylated proteins are radiolabeled via
the phosphorus group. Samples from such cells can then be analyzed
by two-dimensional gels or appropriate immunoassays (such as with
antibodies specific for known phosphoproteins) to detect changes in
protein phosphorylation.
[0081] In a fourth non-limiting example, cells over or under
expressing a gene sequence can be analyzed for the effects on
cellular factors that are not gene products. For instance, the
effect on intracellular concentrations of various small molecules
(such as calcium, sodium, and chloride ions; intermediates in
various enzymatic cycles; lipids; etc.) may be analyzed. In other
instances, the production and expression of various cellular
factors on the cell surface, such as lipids or sugars, are
detected.
[0082] The present invention also provides an advantageous means of
isolating the product encoded by a gene sequence, which can be
simply accomplished by harvesting cells over expressing said
sequence and purifying said product.
[0083] The present invention further provides advantages in that no
functionality need be known for a sequence being over and under
expressed. As such, the time and cost necessary for bioinformatics
may be optionally removed, although the inclusion of bioinformatics
information in the practice of the present invention would increase
the likelihood of accurately assigning functionalities to a gene
sequence.
[0084] Moreover, the invention provides the ability to relate the
functionality of one unidentified gene sequence to another. The
invention further permits the combination of this ability, with the
advantageous capability of identifying functional relatedness
between unidentified and known sequences, to provide the
determination of a family of functionally related gene sequences.
The relatedness of individual family members may be expressed as a
map based on functional relationships, which would otherwise not be
recognized without extensive research.
[0085] All references cited herein, including patents, patent
applications, and publications, are hereby incorporated by
reference in their entireties, whether previously specifically
incorporated or not.
[0086] Having now fully described this invention, it will be
appreciated by those skilled in the art that the same can be
performed within a wide range of equivalent parameters,
concentrations, and conditions without departing from the spirit
and scope of the invention and without undue experimentation.
[0087] While this invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications. This application is intended to
cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth.
* * * * *