U.S. patent application number 13/793961 was filed with the patent office on 2014-01-02 for compositions and methods for the delivery of biologically active rnas.
This patent application is currently assigned to EGEN, INC.. The applicant listed for this patent is EGEN, INC.. Invention is credited to Khursheed Anwer, Jason Fewell, Kevin Polach.
Application Number | 20140005254 13/793961 |
Document ID | / |
Family ID | 42728852 |
Filed Date | 2014-01-02 |
United States Patent
Application |
20140005254 |
Kind Code |
A1 |
Polach; Kevin ; et
al. |
January 2, 2014 |
Compositions and methods for the Delivery of Biologically Active
RNAs
Abstract
The present invention provides novel compounds, compositions,
and methods for the delivery of biologically active RNA molecules
to cells. Specifically, the invention provides novel nucleic acid
molecules, polypeptides, and RNA-protein complexes useful for the
delivery of biologically active RNAs to cells and polynucleotides
encoding the same. The invention also provides vectors for
expressing said polynucleotides. In addition, the invention
provides cells and compositions comprising the novel compounds and
vectors, which can be used as transfection reagents. The invention
further provides methods for producing said compounds, vectors,
cells, and compositions. Additionally, vectors and methods for
delivering biologically active RNA molecules to cells and/or
tissues are provided. The novel compounds, vectors, cells, and
compositions are useful, for example, in delivering biologically
active RNA molecules to cells to modulate target gene expression in
the diagnosis, prevention, amelioration, and/or treatment of
diseases, disorders, or conditions in a subject or organism.
Inventors: |
Polach; Kevin; (Spring Hill,
TN) ; Fewell; Jason; (Madison, AL) ; Anwer;
Khursheed; (Madison, AL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
EGEN, INC.; |
|
|
US |
|
|
Assignee: |
EGEN, INC.
Huntsville
AL
|
Family ID: |
42728852 |
Appl. No.: |
13/793961 |
Filed: |
March 11, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12724408 |
Mar 15, 2010 |
8569065 |
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13793961 |
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61160287 |
Mar 13, 2009 |
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61160288 |
Mar 13, 2009 |
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Current U.S.
Class: |
514/44R ;
435/455; 435/91.3 |
Current CPC
Class: |
C12N 15/63 20130101;
C12N 15/85 20130101; C12N 15/113 20130101; C12N 15/87 20130101 |
Class at
Publication: |
514/44.R ;
435/91.3; 435/455 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Claims
1-36. (canceled)
37. A method for secreting a biologically active RNA from a cell,
comprising: administering to a cell an expression vector comprising
a first polynucleotide that encodes a biologically active RNA
sequence and a recognition RNA sequence, and a second
polynucleotide that encodes a polypeptide comprising an RNA binding
domain that binds the recognition RNA sequence and a non-classical
secretory domain sequence, expressing the first and second
polynucleotides, secreting the biologically active RNA sequence
through a non-classical secretory pathway.
38. The method of claim 37, wherein the cell is a bacterial cell, a
protozoan cell, an insect cell, a yeast cell, a plant cell, a
fungal cell or a mammalian cell.
39. The method of claim 37, wherein biologically active RNA
sequence is selected from the group consisting of an aptamer, a
ribozyme, an antisense nucleic acid, a short interfering RNA
(siRNA), a double stranded RNA (dsRNA), a short hairpin RNA
(shRNA), a micro-RNA (miRNA), and a transcript encoding one or more
biologically active peptides.
40. The method of claim 37, wherein the non-classical secretory
domain sequence nucleotide is selected from the group consisting of
Galectin-1, Galectin-3, Interleukin 1.alpha. (IL-1.alpha.),
Interleukin 1.beta. (IL-1.beta.), Hydrophilic Acylated Surface
Protein B (HASPB), High Mobility Group Box Protein 1 (HMGB1),
Fibroblast Growth Factor 1 (FGF-1), Fibroblast Growth Factor 2
(FGF-2), Interleukin 2 (IL-2), Secretory Transglutaminase,
Annexin-1, Thioredoxin, Rhodanese, and Plasminogen Activator.
41. The method of claim 37, wherein the polypeptide encoded by the
second polynucleotide further comprises a cell penetrating
peptide.
42. The method of claim 41, wherein the cell penetrating peptide
sequence is selected from the group consisting of penetratin,
transportin, model amphiphatic peptide (MAP), trans-activator of
transcription (TAT), homeodomain of antennepedia (AntP), feline
herpes virus (FHV) coat protein, and transportin 10 (TP10).
43. A method for modulating a signaling pathway or the expression
of a target gene of a target cell comprising: administering to a
host cell a vector comprising a first polynucleotide that encodes a
biologically active RNA sequence and a recognition RNA sequence,
and a second polynucleotide that encodes a polypeptide an RNA
binding domain that binds the recognition RNA sequence, a
non-classical secretory domain sequence, and a transport peptide
sequence that facilitates delivery of the biologically active RNA
sequence to neighboring cells and tissues, expressing the vector in
the host cell, secreting the biologically active RNA from the host
cell, delivering the biologically active RNA sequence to a
neighboring cell, wherein the biologically active RNA modulates a
signaling pathway or the expression of a target gene of the target
cell.
44. The method of claim 43, wherein the transport peptide sequence
is a cell penetrating peptide.
45. The method of claim 44, wherein the cell penetrating peptide
sequence is selected from the group consisting of penetratin,
transportin, model amphiphatic peptide (MAP), trans-activator of
transcription (TAT), homeodomain of antennepedia (AntP), feline
herpes virus (FHV) coat protein, and transportin 10 (TP10).
46. The method of claim 43 wherein the host cell and target cell
are in a subject having, suspected of having, or at risk of
developing arthritis, macular degeneration, muscular dystrophy,
Huntington's disease, coronary heart disease, or a cancerous
disease or condition.
47. The method of claim 43, wherein biologically active RNA
sequence is selected from the group consisting of an aptamer, a
ribozyme, an antisense nucleic acid, a short interfering RNA
(siRNA), a double stranded RNA (dsRNA), a short hairpin RNA
(shRNA), a micro-RNA (miRNA), and a transcript encoding one or more
biologically active peptides.
48. The method of claim 43, wherein the non-classical secretory
domain sequence nucleotide is selected from the group consisting of
Galectin-1, Galectin-3, Interleukin 1.alpha. (IL-1.alpha.),
Interleukin 1.beta. (IL-1.beta.), Hydrophilic Acylated Surface
Protein B (HASPB), High Mobility Group Box Protein 1 (HMGB1),
Fibroblast Growth Factor 1 (FGF-1), Fibroblast Growth Factor 2
(FGF-2), Interleukin 2 (IL-2), Secretory Transglutaminase,
Annexin-1, Thioredoxin, Rhodanese, and Plasminogen Activator.
49. The method of claim 37, wherein the host cell and target cell
are in a subject having, suspected of having, or at risk of
developing arthritis and the biologically active RNA sequence is an
Oncostatin M (OSM) inhibitory aptamer.
50. The method of claim 37, wherein the host cell and target cell
are in a subject having, suspected of having, or at risk of
developing macular degeneration and the biologically active RNA
sequence is a Vascular Endothelial Growth Factor (VEGF) inhibitory
aptamer.
51. The method of claim 37, wherein the host cell and target cell
are in a subject having, suspected of having, or at risk of
developing muscular dystrophy and the biologically active RNA
sequence is a Myostatin inhibitory aptamer.
52. The method of claim 37, wherein the host cell and target cell
are in a subject having, suspected of having, or at risk of
developing coronary heart disease and the biologically active RNA
sequence is a von Willebrand factor (vWF) inhibitory aptamer.
53. The method of claim 37, wherein the host cell and target cell
are in a subject having, suspected of having, or at risk of
developing a cancerous disease or condition and the biologically
active RNA sequence is selected from the group consisting of a
Human Epidermal Growth Factor Receptor 3 (HER3) inhibitory aptamer
and a .beta.-catenin inhibitory aptamer.
54. The method of claim 37, wherein the host cell and target cell
are in a subject having, suspected of having, or at risk of
developing arthritis and the biologically active RNA sequence is an
shRNA targeting the Matrix Metalloproteinase-2 (Mmp2)
transcript.
55. The method of claim 37, wherein the host cell and target cell
are in a subject having, suspected of having, or at risk of
developing macular degeneration and the biologically active RNA
sequence is an shRNA targeting the Vascular Endothelial Growth
Factor Receptor (VEGFR) transcript.
56. The method of claim 37, wherein the host cell and target cell
are in a subject having, suspected of having, or at risk of
developing muscular dystrophy and the biologically active RNA
sequence is an shRNA targeting the myostatin transcript.
57. The method of claim 37, wherein the host cell and target cell
are in a subject having, suspected of having, or at risk of
developing Huntington's disease and the biologically active RNA
sequence is an shRNA targeting the huntingtin (HTT) transcript.
58. The method of claim 37, wherein the host cell and target cell
are in a subject having, suspected of having, or at risk of
developing coronary heart disease and the biologically active RNA
sequence is an shRNA targeting the apolipoprotein B (ApoB)
transcript.
59. The method of claim 37, wherein the host cell and target cell
are in a subject having, suspected of having, or at risk of
developing a cancerous disease or condition and the biologically
active RNA sequence is selected from the group consisting of an
shRNA targeting the Epidermal Growth Factor Receptor (EGFR)
transcript, an shRNA targeting the Harvey Rat Sarcoma viral
oncogene homolog (H-Ras) transcript, an shRNA targeting the B-cell
lymphoma-2 (Bcl-2) transcript, an shRNA targeting the surviving
transcript, an shRNA targeting the Focal Adhesion Kinase (FAK)
transcript, an shRNA targeting the Signal Transducer and Activator
of Transcription-3 (STAT-3) transcript, an shRNA targeting the Src
transcript, an shRNA targeting the RET receptor tyrosine kinase
transcript, an shRNA targeting the Kinesin Spindle Protein (KSP)
transcript, and an shRNA targeting the Multiple Drug Resistance
(MDR) transcript.
60. The method of claim 37, wherein the vector further comprises a
first promoter sequence and a termination sequence, and a second
promoter sequence and a polyA addition sequence, wherein the first
polynucleotide is operably linked to the first promoter sequence
and the termination sequence and wherein the second polynucleotide
is operably linked to the second promoter sequence and the polyA
addition sequence
61. The method of claim 43, wherein the vector further comprises a
first promoter sequence and a termination sequence, and a second
promoter sequence and a polyA addition sequence, wherein the first
polynucleotide is operably linked to the first promoter sequence
and the termination sequence and wherein the second polynucleotide
is operably linked to the second promoter sequence and the polyA
addition sequence.
Description
[0001] This application claims priority to U.S. application Ser.
No. 61/160,287, filed Mar. 13, 2009 and U.S. application Ser. No.
61/160,288, filed Mar. 13, 2009, both of which applications are
incorporated by reference herein in their entireties, including the
drawings.
FIELD OF THE INVENTION
[0002] The present invention provides novel compounds,
compositions, and methods for the delivery of biologically active
RNA molecules to cells. Specifically, the invention provides novel
nucleic acid molecules, polypeptides, and RNA-protein complexes
useful for the delivery of biologically active RNAs to cells and
polynucleotides encoding the same. The invention also provides
vectors for expressing said polynucleotides. In addition, the
invention provides cells and compositions comprising the novel
compounds and vectors, which can be used as transfection reagents,
among other things. The invention further provides methods for
producing said compounds, vectors, cells, and compositions.
Additionally, vectors and methods for delivering biologically
active RNA molecules, such as ribozymes, antisense nucleic acids,
allozymes, aptamers, short interfering RNA (siRNA), double-stranded
RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA)
molecules, to cells and/or tissues are provided. The novel
compounds, vectors, cells, and compositions are useful, for
example, in delivering biologically active RNA molecules to cells
to modulate target gene expression in the diagnosis, prevention,
amelioration, and/or treatment of diseases, disorders, or
conditions in a subject or organism.
BACKGROUND OF THE INVENTION
[0003] RNA molecules have the capacity to act as potent modulators
of gene expression in vitro and in vivo. These molecules can
function through a number of mechanisms utilizing either specific
interactions with cellular proteins or base pairing interactions
with other RNA molecules. This modulation can act in opposition to
the cellular machinery, as with RNA aptamers that disrupt
RNA-protein and protein-protein interactions, or in concert with
cellular processes, as with siRNAs that act by redirecting the
endogenous RNAi machinery to targets of choice. Modulation of gene
expression via RNA effector molecules has great therapeutic
potential as the modulatory complexes formed, be they RNA-protein
complexes or RNA-RNA complexes, are often highly specific (Aagaard
et al., 2007, Adv Drug Deliv Rev., 59:75-86; de Fougerolles et al.,
2007, Nat Rev Drug Discov., 6:443-53; Grimm et al., 2007, J Clin
Invest., 117:3633-411-4; Rayburn et al., 2008, Drug Discov Today.,
13:513-21). When this specificity is determined by the well
established rules of base pairing, targeting of this regulatory
machinery to particular gene products becomes accessible to direct
experimental design.
[0004] RNA molecules that modulate gene expression may take a
number of different forms. Perhaps the seminal example for all is
the antisense RNA molecule. This inhibitory RNA is typically a
direct complement of the mRNA transcript it targets and functions
by presenting an obstacle to the translational machinery and also
by targeting the transcript for degradation by cellular nucleases.
Another related and overlapping class is the small inhibitory RNA
(siRNA) which acts through the post-transcriptional gene silencing
or RNAi pathway. These RNAs are typically about 21-23 nucleotides
in length and associate with specific cellular proteins to form
RNA-induced silencing complexes (RISCs). These small RNAs are also
complementary to sequences within their mRNA targets and binding of
these complexes leads to translational silencing or degradation of
the transcripts (Farazi et al., 2008, Development., 135:1201-145-7;
Sontheimer et al., 2005, Nat Rev Mol Cell Biol., 6:127-38; Zamore
et al., 2005, Science., 309:1519-24).
[0005] Two additional classes of RNA molecules that can modulate
gene expression and activity are the catalytic RNA ribozymes and
the competitive RNA aptamers. Ribozymes are RNA based enzymes that
catalyze chemical reactions on RNA substrates, most often
hydrolysis of the phosphodiester backbone. Formation of the
catalytic active site requires base pairing between the ribozyme
and the RNA substrate, so ribozyme activity can also be targeted to
desired substrates by providing appropriate guide sequences (Wood
et al., 2007, PLoS Genet., 3:e109; Scherer et al., 2007, Gene
Ther., 14:1057-64; Trang et al., 2004, Cell Microbiol., 6:499-508).
When targeted to mRNA transcripts, ribozymes have the potential to
cleave those transcripts and lead to downregulation of the
associated protein (Liu et al., 2007, Cancer Biol Ther., 6:697-704;
Song et al., 2008, Cancer Gene Ther.; Weng et al., 2005, Mol Cancer
Ther., 4:948-55; Li et al., 2005, Mol. Ther. 12:900-9). RNA
aptamers are typically selected from pools of random RNA sequences
by their ability to interact with a target molecule, often a
protein molecule. Engineering RNA aptamers is less straightforward
as the binding is not defined by base pairing interactions, but
once an effective sequence is found the specificity and affinity of
the binding often rivals that of antibody-antigen interactions (Mi
et al., 2008, Mol. Ther., 16:66-73; Lee et al., 2007, Cancer Res.,
67:9315-21; Ireson et al., 2006, Mol Cancer Ther., 12:2957-62;
Cerchia et al., 2005, PLoS Biol., 3:e1230). RNA aptamers also have
a greater range of target molecules and the potential to alter gene
activity via a number of different mechanisms.
[0006] Two methods for delivering inhibitory RNA molecules to cells
have become standard practice. The first method involves production
of the RNA molecules in the test tube by using purified polymerases
and DNA templates or through direct chemical synthesis. These RNA
molecules can then be purified and mixed with a synthetic carrier,
typically a polymer, a liposome, or a peptide, and delivered to the
target cells (Aigner et al., 2007, Appl Microbiol Biotechnol.,
76:9-21; Juliano et al., 2008, Nucleic Acids Res., 36:4158-71;
Akhtar et al., 2007, J Clin Invest., 117:3623-32). These molecules
are delivered to the cytoplasm where they bind to their mRNA or
protein targets directly or through the formation of modulatory
complexes. The second method involves transfecting the target cells
with a plasmid molecule encoding the biologically active RNA. Once
again, the purified plasmid molecule is coupled with a synthetic
carrier in the test tube and delivered to the target cell (Fewell
et al., 2005, J Control Release., 109:288-98; Wolff et al., 2008,
Mol. Ther., 16:8-15; Gary et al., 2007, J Control Release.,
121:64-73).
[0007] In this case, the plasmid template must be delivered to the
cell nucleus where the DNA is transcribed into the biologically
active RNA molecule. This RNA is then exported to the cytoplasm,
where it fords its way to modulatory complexes and specific mRNA
transcript targets. With each of these approaches, the extent of
gene regulation within a population of cells is limited by the
transfection efficiency of the delivery system. Cells that are not
transfected with the biologically active RNA molecules or plasmids
encoding biologically active RNAs have no mechanism for receiving
the modulatory signal. Although high transfection efficiencies are
possible for cells growing in culture, achieving similar extents of
transfection is difficult in vivo. This delivery issue is currently
the major prohibitive factor for the application of RNA based
therapeutics in vivo as it limits the extent to which a particular
gene can be regulated in a population of cells. Thus, there remains
a need to for an effective delivery system for efficiently
delivering biologically active RNAs to cells and tissues.
SUMMARY OF THE INVENTION
[0008] The present invention provides novel approaches for
circumventing the current problems associated with low transfection
efficiencies in the delivery of biologically active RNA molecules
to mammalian cells and tissues. One approach involves the use of
one or more "bioreactor" cells which produce and subsequently
secrete one or more biologically active RNA molecules, such as
ribozymes, antisense nucleic acids, allozymes, aptamers, short
interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA
(miRNA), and short hairpin RNA (shRNA) molecules, as well as RNA
transcripts encoding one or more biologically active peptides,
thereby delivering said molecule(s) to the extracellular matrix and
surrounding cells and tissues. The bioreactor cell is generated by
administering to a cell one or more expression vectors designed to
produce an RNA-protein complex comprising at least one biologically
active RNA molecule targeting one or more genes of interest and a
fusion protein capable of delivering the biologically active RNA
molecule(s) to the extracellular matrix and/or neighboring cells
and tissues.
[0009] The RNA portion of the RNA-protein complex comprises at
least a recognition RNA sequence and one or more biologically
active RNA sequences. The protein portion of the RNA-protein
complex is a fusion protein comprising at least an RNA binding
domain and a transport peptide. Examples of suitable transport
peptides include, but are not limited to, one or more peptides
selected from a cell penetrating peptide, a non-classical secretory
domain, an endosomal release domain, a receptor binding domain, and
a fusogenic peptide. The RNA portion and the protein portion of the
RNA-protein complex are expressed from one or more vectors in the
nucleus of the transfected bioreactor cell and are transported to
the cytoplasm, where the fusion protein is translated and binds to
the RNA sequence comprising the biologically active RNA, thereby
generating the RNA-protein complex. The RNA-protein complex is
secreted from the bioreactor cell and remains intact in the
extracellular matrix. The RNA-protein complex can remain in the
extracellular matrix where it exerts its modulatory action within
the extracellular matrix or at the cell surface of a neighboring
target cells. Alternatively, the RNA-protein complex can be
designed such that, at the surface of a target cell, the fusion
protein facilitates import of the biologically active RNA to the
cytoplasm of the target cell.
[0010] Thus, in essence, the transfected cells are converted into
"bioreactors" that produce and deliver biologically active RNA
molecules, secreted as RNA-protein complexes, to the extracellular
matrix and/or other neighboring cells. This approach takes
advantage of the amplification of the modulatory signal provided by
directing the cellular machinery to synthesize the biologically
active RNA molecules from the plasmid template. Thus, the
modulatory signal is no longer bound by the initial transfection
efficiency of a single delivery event but has the potential to
reach many cells over a sustained period of time.
[0011] Such bioreactor cells can also be generated in cell culture
by transfection of appropriate cells with one or more of the
expression vectors described herein. In essence, the transfected
cells are converted into bioreactors that produce and deliver the
biologically active RNA molecules to other cells in culture.
Accordingly, the bioreactor cells have in vivo and ex vivo
applications as a therapeutic delivery system, as well as in vitro
and in vivo applications as a novel transfection agent.
[0012] The purpose of the bioreactor cell is to secrete a
biologically active RNA molecule to the extracellular matrix in a
form that can then function within the extracellular matrix or at
the cell surface of a neighboring target cells or can be delivered
to neighboring target cells. Viral packaging cells can serve the
same purpose: secretion and delivery of biologically active RNA
molecules. But in contrast to the bioreactor producing fusion
proteins which are assembled from individual domains taken from
various sources, the viral particles have evolved for the purpose
of transferring nucleic acids from one cell to another (thus,
mobile genetic elements). Both RNA and DNA viruses can be utilized
as potential carriers for nucleic acid modulators. In the case of
RNA viruses, a polynucleotide encoding the biologically active RNA
molecule is added to a viral transcript encoding the non-structural
genes of the virus. This transcript serves both as template for the
viral proteins responsible for viral processes and as the genome
which is packaged into the viral particles. The biologically active
RNA is coupled with the RNA encoding the non-structural genes so
that the biologically active RNA is incorporated into the virus
particles. In the case of DNA viruses, a DNA segment encoding the
biologically active RNA is added to the viral DNA such that
synthesis of the viral transcript produces the biologically active
RNA as well. The viral particles are assembled from the structural
proteins encoded by transcripts produced from the helper plasmid.
Likewise, one or more polynucleotides encoding the biologically
active RNA and the fusion protein can be added to a viral
transcript encoding the non-structural viral genes (in the case of
RNA viruses) or added to the viral DNA (in the case of DNA
viruses). Thus, cells transfected with expression vectors
comprising sequence for encoding viral non-structural genes and
sequence for encoding either a biologically active RNA or a
biologically active RNA-protein complex of the invention can be
used in the same manner as the bioreactor cells, as described
herein.
[0013] These approaches directly address the key issue in
application of plasmid based RNA-mediated therapeutics, namely the
low transfection efficiencies associated with plasmid delivery. Use
of the described bioreactor cells circumvents the need for high
efficiency transfection, as the RNA-mediated effect is amplified
through the in vivo production and delivery of biologically active
RNAs to surrounding cells and tissues.
[0014] The present invention thus provides novel nucleic acid
molecules, polypeptides, RNA-protein complexes, polynucleotides,
and vectors useful for the delivery of biologically active RNA
molecules to mammalian cells and tissues. In addition, the
invention provides compositions comprising said nucleic acid
molecules, polypeptides, RNA-protein complexes, polynucleotides and
vectors. The invention also provides cells comprising the nucleic
acid molecules, polypeptides, RNA-protein complexes,
polynucleotides and vectors of the invention. Additionally, the
invention provides methods of producing the nucleic acid molecules,
polypeptides, RNA-protein complexes, polynucleotides, vectors,
compositions, and cells of the invention, as well as therapeutic
methods for using the inventive molecules in vitro, ex vivo, and in
vivo.
[0015] The present invention provides novel expression vectors
useful in the production of the nucleic acid molecules,
polypeptides, and RNA-protein complexes of the invention. In one
embodiment, the invention provides an expression vector that
expresses an RNA-protein complex of the invention. Thus, in one
embodiment, the invention provides an expression vector comprising
a polynucleotide that encodes a nucleic acid comprising one or more
biologically active RNA sequences, a recognition RNA sequence, and
optionally a terminal minihelix sequence, and a polynucleotide that
encodes a polypeptide comprising an RNA binding domain and one or
more transport peptides. The RNA portion and the protein portion of
the RNA-protein complex expressed from the expression vector are
expressed in the nucleus of the transfected bioreactor cell and are
transported separately to the cytoplasm, where the fusion protein
is translated and binds to the RNA sequence comprising the
biologically active RNA, thereby generating the RNA-protein
complex. The RNA-protein complex is secreted from the bioreactor
cell as discussed herein. The one or more biologically active RNA
sequences can be one or more different types of biologically active
RNA sequences directed to the same gene target or can be
biologically active RNA sequences directed to different gene
targets.
[0016] In a further embodiment, the expression vector additionally
comprises a first promoter sequence, a termination sequence, and
optionally one or more primers sequences, a second promoter
sequence, a polyA addition sequence, and optionally one or more
primers sequences, wherein the polynucleotide encoding the first
biologically active RNA sequence, the recognition RNA sequence, and
the optional terminal minihelix sequence is operably linked to the
first promoter sequence and the termination sequence and wherein
the polynucleotide encoding the RNA binding domain sequence and the
transport peptide sequence is operably linked to the second
promoter sequence and the polyA addition sequence.
[0017] In another embodiment, the expression vector further
comprises one or more polynucleotide sequences encoding one or more
viral polymerases and one or more viral accessory proteins
necessary for viral replication. In a further embodiment, the
vector additionally comprises one or more promoter sequences, one
or more polyA addition sequences, and optionally one or more
primers sequences, wherein the polynucleotide sequence(s) encoding
the viral polymerase(s) and the viral accessory protein(s) is
operably linked to the one or more promoter sequences and the one
or more polyA addition sequences. The vectors comprising viral
polymerase and accessory protein sequences can be used with
expression vectors comprising one or more polynucleotide sequences
encoding one or more viral coat proteins and one or more viral
fusogenic proteins. In a further embodiment, the expression vectors
comprising one or more polynucleotide sequences encoding one or
more viral coat proteins and one or more viral fusogenic proteins
can further comprise one or more promoter sequences and one or more
polyA addition sequences, wherein the polynucleotide sequence(s)
encoding the viral coat protein(s) and the viral fusogenic
protein(s) is operably linked to the one or more promoter sequences
and the one or more polyA addition sequences.
[0018] In certain embodiments of the described expression vectors,
the biologically active RNA sequence is selected from a ribozyme,
antisense nucleic acid, allozyme, aptamer, short interfering RNA
(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short
hairpin RNA (shRNA), and a transcript encoding one or more
biologically active peptides. In one specific embodiment, the
biologically active RNA sequence is a short hairpin RNA (shRNA). In
another specific embodiment, the biologically active RNA sequence
is an aptamer. In certain embodiments, the recognition RNA sequence
is selected from a U1 loop, Group II intron, NRE stem loop, S1A
stem loop, Bacteriophage BoxBR, HIV Rev response element, AMVCP
recognition sequence, and ARE sequence. In one embodiment, the
terminal minihelix sequence is from the adenovirus VA1 RNA
molecule. In certain embodiments, the RNA binding domain is
selected from a U1A, CRS1, CRM1, Nucleolin RBD12, hRBMY,
Bacteriophage Protein N, HIV Rev, alfalfa mosaic virus coat protein
(AMVCP), and tristetrapolin amino acid sequence. In certain
embodiments, the one or more transport peptides is selected from a
cell penetrating peptide, a non-classical secretory domain, a
receptor binding domain, a fusogenic peptide, and an endosomal
release domain, as well as any combinations thereof. In one
specific embodiment, the transport peptide is a cell penetrating
peptide. In certain specific embodiments, the cell penetrating
peptide is selected from a penetratin, transportan, MAP, HIV TAT,
Antp, Rev, FHV coat protein, TP10, and pVEC sequence. In another
specific embodiment, the transport peptide is a non-classical
secretory domain. In certain specific embodiments, the
non-classical secretory domain is selected from a Galcetin-1
peptide, Galectin-3 peptide, IL-1.alpha., IL-1.beta., HASPB, HMGB1,
FGF-1, FGF-2, IL-2 signal, secretory transglutaminase, annexin-1,
HTV TAT, Herpes VP22, thioredoxin, Rhodanese, and plasminogen
activator signal sequence. In one specific embodiment, the
transport peptides are a cell penetrating peptide, and one or more
transport peptides selected from a non-classical secretory domain,
a receptor binding domain, a fusogenic peptide, and an endosomal
release domain. In one specific embodiment, the transport peptides
are a cell penetrating peptide, and a non-classical secretory
domain. In certain embodiments, the viral non-structural and
structural genes (viral polymerases, accessory proteins, coat
proteins, and fusogenic proteins) are selected from DNA viruses and
RNA viruses, including, but not limited to, Adenovirus,
Adeno-Associated Virus, Herpes Simplex Virus Lentivirus,
Retrovirus, Sindbis virus, and Foamy virus.
[0019] In any of the above-described embodiments, the expression
vector can further comprise an additional polynucleotide sequence
that encodes a nucleic acid comprising one or more biologically
active RNA sequences that target one or more further gene
target(s). In one embodiment, the additional polynucleotide
sequence encodes a nucleic acid comprising one or more biologically
active RNA sequences that target a further gene target and an RNA
recognition sequence. In another embodiment, where one of the
biologically active RNA sequences in the vector is a short
interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA
(miRNA), or short hairpin RNA (shRNA), the expression vector
additionally comprises a polynucleotide that encodes a nucleic acid
comprising one or more biologically active RNA sequences targeted
to Dicer and/or Drosha. None of the polynucleotide sequences
encoding nucleic acid comprising one or more biologically active
RNA sequences targeted to Dicer and/or Drosha comprise a
recognition RNA sequence.
[0020] In another embodiment, the expression vector comprises a
first expression cassette and a second expression cassette, wherein
the first expression cassette comprises a promoter sequence, one or
biologically active RNA sequences directed to one or more target
genes, a recognition RNA sequence, optionally a terminal minihelix
sequence, a termination sequence, and optionally one or more
primers sequences, wherein the biologically active RNA sequence(s),
the recognition RNA sequence, and the optional terminal minihelix
sequence are operably linked to the promoter sequence and the
termination sequence; and the second expression cassette comprises
a promoter sequence, an RNA binding domain sequence, a transport
peptide sequence, a poly A addition sequence, and optionally one or
more primers sequences, wherein the RNA binding domain sequence and
the transport peptide sequence are operably linked to the promoter
sequence and the poly A addition sequence. In a further embodiment,
the expression vector additionally comprises a third expression
cassette, wherein the third expression cassette comprises one or
more promoter sequences, one or more polynucleotide sequences
encoding one or more viral polymerases and one or more viral
accessory proteins necessary for viral replication, one or more
polyA addition sequences, and optionally one or more primers
sequences, wherein the polynucleotide sequence(s) encoding the
viral polymerase(s) and the viral accessory protein(s) is operably
linked to the one or more promoter sequences and the one or more
polyA addition sequences. The vectors comprising a third expression
cassette comprising viral polymerase and accessory protein
sequences can be used with expression vectors comprising one or
more polynucleotide sequences encoding one or more viral coat
proteins and one or more viral fusogenic proteins. In a further
embodiment, the expression vectors comprising one or more
polynucleotide sequences encoding one or more viral coat proteins
and one or more viral fusogenic proteins can further comprise one
or more promoter sequences and one or more polyA addition
sequences, wherein the polynucleotide sequence(s) encoding the
viral coat protein(s) and the viral fusogenic protein(s) is
operably linked to the one or more promoter sequences and the one
or more polyA addition sequences.
[0021] In one embodiment of the above-described expression vectors,
the expression cassette comprising the RNA portion of the
RNA-protein complex, (i.e., comprising an RNA recognition sequence,
one or more biologically active RNAs and optionally a terminal
minihelix sequence) is ligated into an artificial intron within the
expression cassette for the fusion protein (i.e., RNA binding
domain and one or more transport peptides). In this expression
vector, the Sec-RNA is encoded within an artificial intron placed
within the mRNA sequence encoding the fusion protein. DNA fragments
encoding for Sec-RNA molecules or fusion proteins are prepared by
PCR. DNA fragments encoding for Sec-RNA molecules are prepared with
primers including splice donor and acceptor sites and restriction
sites for subcloning into a unique restriction site within the
fusion protein sequence. DNA fragments encoding for the fusion
protein are prepared with primers including restriction sites for
subcloning into the plasmids described above. After transcription,
the Sec-RNA is released from the mRNA encoding the fusion protein
by the splicing machinery endogenous to the bioreactor cell.
[0022] In any of these embodiments, the biologically active RNA
sequence is selected from a ribozyme, antisense nucleic acid,
allozyme, aptamer, short interfering RNA (siRNA), double-stranded
RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), and a
transcript encoding one or more biologically active peptides. The
recognition RNA sequence is selected from a U1 loop, Group II
intron, NRE stem loop, S1A stem loop, Bacteriophage BoxBR, HIV Rev
response element, AMVCP recognition sequence, and ARE sequence. The
terminal minihelix sequence is selected from the adenovirus VA1 RNA
molecule. The RNA binding domain is selected from a U1A, CRS1,
CRM1, Nucleolin RBD12, hRBMY, Bacteriophage Protein N, HIV Rev,
alfalfa mosaic virus coat protein (AMVCP), and tristetrapolin amino
acid sequence. The one or more transport peptides is selected from
a cell penetrating peptide, a non-classical secretory domain, a
receptor binding domain, a fusogenic peptide, and an endosomal
release domain, as well as any combinations thereof.
[0023] In any of the above-described embodiments, the expression
vector can further comprise an additional expression cassette,
wherein the additional expression cassette comprises one or more
promoter sequences, one or more polynucleotide sequences encoding
nucleic acid comprising one or more biologically active RNA
sequences that target a further gene transcript and one or more
polyA addition sequences, wherein the polynucleotide sequence
encoding nucleic acid comprising one or more biologically active
RNA sequences that target a further gene transcript is operably
linked to the one or more promoter sequences and the one or more
polyA addition sequences. In one embodiment, the additional
polynucleotide sequence encodes a nucleic acid comprising one or
more biologically active RNA sequences that target a further gene
transcript and an RNA recognition sequence. In another embodiment,
where one of the biologically active RNA sequences in the vector is
a short interfering RNA (siRNA), double-stranded RNA (dsRNA),
micro-RNA (miRNA), or short hairpin RNA (shRNA), the additional
polynucleotide sequence encodes nucleic acid comprising one or more
biologically active RNA sequences targeted to Dicer and/or Drosha.
None of the polynucleotide sequences encoding nucleic acid
comprising one or more biologically active RNA sequences targeted
to Dicer and/or Drosha comprise a recognition RNA sequence.
[0024] In one embodiment, the invention provides an expression
vector comprising a polynucleotide that encodes a nucleic acid
molecule comprising one or more biologically active RNA sequences,
a recognition RNA sequence, and an optional terminal minihelix
sequence. In one embodiment, the expression vector comprises a
polynucleotide that encodes a nucleic acid molecule comprising one
or more biologically active RNA sequences and one or more
polynucleotide sequences encoding one or more viral polymerases and
one or more viral accessory proteins necessary for viral
replication. In certain embodiments, the expression vector
comprises a polynucleotide encoding a nucleic acid molecule wherein
the biologically active RNA sequence is selected from a ribozyme,
antisense nucleic acid, allozyme, aptamer, short interfering RNA
(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short
hairpin RNA (shRNA), and a transcript encoding one or more
biologically active peptides. In one specific embodiment, the
expression vector comprises a polynucleotide encoding a nucleic
acid molecule wherein the biologically active RNA sequence is a
short hairpin RNA (shRNA). In another specific embodiment, the
expression vector comprises a polynucleotide encoding a nucleic
acid molecule wherein the biologically active RNA sequence is an
aptamer. In certain embodiments, the expression vector comprises a
polynucleotide encoding a nucleic acid molecule wherein the
recognition RNA sequence is selected from a U1 loop, Group II
intron, NRE stem loop, S1A stem loop, Bacteriophage BoxBR, HIV Rev
response element, AMVCP recognition sequence, and ARE sequence. In
one embodiment, the terminal minihelix sequence is from the
adenovirus VA1 RNA molecule.
[0025] The invention also provides an expression vector comprising
a polynucleotide that encodes a polypeptide comprising an RNA
binding domain and one or more transport peptides. In certain
embodiments, the RNA binding domain is selected from a U1A, CRS1,
CRM1, Nucleolin RBD12, hRBMY, Bacteriophage Protein N, HIV Rev,
alfalfa mosaic virus coat protein (AMVCP), and tristetrapolin amino
acid sequence. In certain embodiments, the one or more transport
peptides is selected from a cell penetrating peptide, a
non-classical secretory domain, a receptor binding domain, a
fusogenic peptide, and an endosomal release domain, as well as any
combinations thereof. In one embodiment, the invention provides an
expression vector comprising a polynucleotide that encodes a
polypeptide comprising an RNA binding domain and a cell penetrating
peptide. In certain specific embodiments, the cell penetrating
peptide is selected from a penetratin, transportan, MAP, HIV TAT,
Antp, Rev, FHV coat protein, TP10, and pVEC sequence. In another
embodiment, the invention provides an expression vector comprising
a polynucleotide that encodes a polypeptide comprising an RNA
binding domain and a non-classical secretory domain. In certain
specific embodiments, the non-classical secretory domain is
selected from a Galcetin-1 peptide, Galectin-3 peptide,
IL-1.alpha., IL-1.beta., HASPB, HMGB1, FGF-1, FGF-2, IL-2 signal,
secretory transglutaminase, annexin-1, HIV TAT, Herpes VP22,
thioredoxin, Rhodanese, and plasminogen activator signal sequence.
In one embodiment, the invention provides an expression vector
comprising a polynucleotide that encodes a polypeptide comprising
an RNA binding domain, a cell penetrating peptide, and one or more
transport peptides selected from a non-classical secretory domain,
a receptor binding domain, a fusogenic peptide, and an endosomal
release domain. In one embodiment, the invention provides an
expression vector comprising a polynucleotide that encodes a
polypeptide comprising an RNA binding domain, a cell penetrating
peptide, and a non-classical secretory domain.
[0026] Thus, the invention provides a first expression vector
comprising a polynucleotide that encodes a nucleic acid molecule
comprising one or more biologically active RNA sequences, a
recognition RNA sequence and optionally a terminal minihelix
sequence and a second expression vector comprising a polynucleotide
that encodes a polypeptide comprising an RNA binding domain and one
or more transport peptides, for example, a peptide selected from a
cell penetrating peptide, a non-classical secretory domain, a
receptor binding domain, a fusogenic peptide, and an endosomal
release domain. The RNA portion of the RNA-protein complex
expressed from the first expression vector and the protein portion
of the RNA-protein complex expressed from the second expression
vector are expressed in the nucleus of the transfected bioreactor
cell and are transported separately to the cytoplasm, where the
fusion protein is translated and binds to the RNA sequence
comprising the biologically active RNA, thereby generating the
RNA-protein complex. The RNA-protein complex is secreted from the
bioreactor cell as discussed herein.
[0027] In any of the expression vectors of the invention, one or
more of the sequences comprising the recognition RNA sequence, the
individual biologically active RNA sequences, the optional terminal
minihelix sequence, the RNA binding domain, and the transport
peptide(s), as well as any other sequences, including viral
sequences, promoters, primers, termination sequences, and polyA
sequences are joined directly without the addition of one or more
intervening or additional sequences. Alternatively, one or more of
the sequences comprising the recognition RNA sequence, the
individual biologically active RNA sequences, the optional terminal
minihelix sequence, the RNA binding domain, and the transport
peptide(s), as well as any other sequences, including viral
sequences, promoters, primers, termination sequences, and polyA
sequences are joined with the addition of one or more intervening
or additional sequences. In any of the above-described embodiments,
the individual biologically active RNA sequences themselves are
joined directly without any intervening or additional sequences or
are joined with the addition of one or more intervening or
additional sequences. In any of the above-described embodiments,
the recognition RNA sequence and any of the biologically active
RNAs are joined directly without the addition of one or more
linker, spacer, or other sequences or are joined with the addition
of one or more linker, spacer, and/or other sequences. In any of
the above-described embodiments, the RNA binding domain and any of
the individual transport peptides are joined directly without the
addition of one or more linker, spacer, or other sequences or are
joined with the addition of one or more linker, spacer, and/or
other sequences.
[0028] In any of the expression vectors of the invention, the
vector is selected from a suitable backbone vector. Examples of
suitable vectors include those derived from pCI, pET, pSI, pcDNA,
pCMV, etc. In certain embodiments, the vector is selected from
pEGEN 1.1, pEGEN 2.1, pEGEN 3.1, and pEGEN 4.1. The pEGEN vectors
are derived from pSI (Promega, product #E1721), pCI (Promega,
product #E1731), pVAX (Invitrogen, product #12727-010) and other in
house constructs. In one embodiment, the vector comprises a pUC
origin of replication. In one embodiment, the expression vector
comprises a drug resistance gene. Non-limiting examples of suitable
drug resistance genes include those selected from puromycin,
ampicillin, tetracycline, and chloramphenicol resistant genes, as
well as any other drug resistant genes known and described in the
art.
[0029] The invention also provides compositions comprising one or
more expression vectors of the invention and a pharmaceutically
acceptable carrier. The expression vector of the composition can be
any of the expression vectors described herein. In one embodiment,
the composition comprises an expression vector comprising a
polynucleotide encoding a nucleic acid comprising one or more
biologically active RNA sequences, a recognition RNA sequence,
optionally a terminal minihelix sequence, and a polynucleotide
encoding a polypeptide comprising an RNA binding domain, and one or
more transport peptide sequences (for example, a cell penetrating
peptide, non-classical secretory domain, endosomal release domain,
receptor binding domain, fusogenic peptide) and a pharmaceutically
acceptable carrier. In one embodiment, the composition further
comprises a second expression vector comprising a polynucleotide
sequence that encodes a nucleic acid comprising one or more
biologically active RNA sequences that target one or more further
gene target(s). In one embodiment, the additional polynucleotide
sequence encodes a nucleic acid comprising one or more biologically
active RNA sequences that target one or more further gene targets
and an RNA recognition sequence. In another embodiment, where one
of the biologically active RNA sequences in the vector is a short
interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA
(miRNA), or short hairpin RNA (shRNA), the expression vector
additionally comprises a polynucleotide that encodes a nucleic acid
comprising one or more biologically active RNA sequences targeted
to Dicer and/or Drosha.
[0030] In one embodiment, the composition comprises an expression
vector comprising a polynucleotide sequence encoding a nucleic acid
comprising one or more biologically active RNA sequences, a
recognition RNA sequence, optionally a terminal minihelix sequence,
and a polynucleotide sequence encoding a polypeptide comprising an
RNA binding domain, and one or more transport peptide sequences
(for example, a cell penetrating peptide, non-classical secretory
domain, endosomal release domain, receptor binding domain,
fusogenic peptide) and a polynucleotide sequence encoding a nucleic
acid comprising one or more biologically active RNA sequences that
target Dicer and/or Drosha and a pharmaceutically acceptable
carrier.
[0031] In one embodiment, the composition comprises an expression
vector comprising a polynucleotide encoding a nucleic acid
comprising one or more biologically active RNA sequences, a
recognition RNA sequence, optionally a terminal minihelix sequence,
and a polynucleotide encoding a polypeptide comprising an RNA
binding domain, and one or more transport peptide sequences, as
well as a first promoter sequence, a termination sequence, and
optionally one or more primers sequences, a second promoter
sequence, a polyA addition sequence, and optionally one or more
primers sequences and a pharmaceutically acceptable carrier. In
this embodiment, the polynucleotide encoding the first biologically
active RNA sequence, the recognition RNA sequence, and the optional
terminal minihelix sequence is operably linked to the first
promoter sequence and the termination sequence and the
polynucleotide encoding the RNA binding domain sequence and the
transport peptide sequence is operably linked to the second
promoter sequence and the polyA addition sequence.
[0032] In one embodiment, the composition comprises an expression
vector comprising a polynucleotide encoding a nucleic acid
comprising one or more biologically active RNA sequences, a
recognition RNA sequence, optionally a terminal minihelix sequence,
a polynucleotide encoding a polypeptide comprising an RNA binding
domain, and one or more transport peptide sequences, and a
polynucleotide encoding a nucleic acid comprising one or more
biologically active RNA sequences targeted to Dicer and/or Drosha,
as well as a first promoter sequence, a first termination sequence,
and optionally one or more primers sequences, a second promoter
sequence, a polyA addition sequence, and optionally one or more
primer sequences, and a one or more further promoter sequences, one
or more further termination sequences, and one or more primer
sequences and a pharmaceutically acceptable carrier. In this
embodiment, the polynucleotide encoding the first biologically
active RNA sequence, the recognition RNA sequence, and the optional
terminal minihelix sequence is operably linked to the first
promoter sequence and the first termination sequence and the
polynucleotide encoding the RNA binding domain sequence and the
transport peptide sequence is operably linked to the second
promoter sequence and the polyA addition sequence and the
polynucleotide encoding the one or more biologically active RNA
sequences targeted to Dicer and/or Drosha is operably linked to the
one or more further promoter sequence and the one or more further
termination sequences.
[0033] In one embodiment, the composition comprises a first
expression vector comprising a polynucleotide encoding a nucleic
acid comprising one or more biologically active RNA sequences, a
recognition RNA sequence, optionally a terminal minihelix sequence,
and a polynucleotide encoding a polypeptide comprising an RNA
binding domain, and one or more transport peptide sequences, and
one or more polynucleotide sequences encoding one or more viral
polymerases and one or more viral accessory proteins necessary for
viral replication and a second expression vector comprising one or
more polynucleotide sequences encoding one or more viral coat
proteins and one or more viral fusogenic proteins in a
pharmaceutically acceptable carrier. In certain embodiments, the
expression vectors of these compositions additionally comprise a
first promoter sequence, a termination sequence, and optionally one
or more primer sequences, a second promoter sequence, a polyA
addition sequence, and optionally one or more primer sequences, and
a one or more further promoter sequences, one or more further polyA
addition sequences, and optionally one or more further primers
sequences, wherein the polynucleotide encoding the first
biologically active RNA sequence, the recognition RNA sequence, and
the optional terminal minihelix sequence is operably linked to the
first promoter sequence and the termination sequence and wherein
the polynucleotide encoding the RNA binding domain sequence and the
transport peptide sequence is operably linked to the second
promoter sequence and the polyA addition sequence, and wherein the
one or more polynucleotides encoding one or more viral polymerases
and one or more viral accessory proteins are operably linked to the
one or more promoter sequences and one or more polyA addition
sequences, and wherein the one or more polynucleotide sequences
encoding the viral coat protein(s) and the viral fusogenic
protein(s) are operably linked to the one or more promoter
sequences and the one or more polyA addition sequences.
[0034] In one embodiment, the composition comprises an expression
vector comprising a polynucleotide that encodes a nucleic acid
molecule comprising one or more biologically active RNA sequences,
a recognition RNA sequence, and optionally a terminal minihelix
sequence and a pharmaceutically acceptable carrier.
[0035] In one embodiment, the composition comprises an expression
vector comprising a polynucleotide that encodes a polypeptide
comprising an RNA binding domain and one or more transport peptide
sequences (for example, a cell penetrating peptide, non-classical
secretory domain, endosomal release domain, receptor binding
domain, fusogenic peptide) and a pharmaceutically acceptable
carrier.
[0036] In one embodiment, the composition comprises a first
expression vector comprising a polynucleotide that encodes a
nucleic acid molecule comprising one or more biologically active
RNA sequences, a recognition RNA sequence, and optionally a
terminal minihelix sequence and a second expression vector
comprising a polynucleotide that encodes a polypeptide comprising
an RNA binding domain and one or more transport peptide sequences
(for example, a cell penetrating peptide, non-classical secretory
domain, endosomal release domain, receptor binding domain,
fusogenic peptide) and a pharmaceutically acceptable carrier. In
one embodiment, the composition further comprises a third
expression vector comprising a polynucleotide sequence that encodes
a nucleic acid comprising one or more biologically active RNA
sequences that target one or more further gene target(s). In one
embodiment, the additional polynucleotide sequence encodes a
nucleic acid comprising one or more biologically active RNA
sequences that target a further gene target and an RNA recognition
sequence. In another embodiment, where one of the biologically
active RNA sequences in the vector is a short interfering RNA
(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), or short
hairpin RNA (shRNA), the expression vector additionally comprises a
polynucleotide that encodes a nucleic acid comprising one or more
biologically active RNA sequences targeted to Dicer and/or
Drosha.
[0037] In one embodiment, the composition comprises a first
expression vector comprising a polynucleotide encoding a nucleic
acid comprising one or more biologically active RNA sequences and
one or more polynucleotide sequences encoding one or more viral
polymerases and one or more viral accessory proteins necessary for
viral replication and a second expression vector comprising one or
more polynucleotide sequences encoding one or more viral coat
proteins and one or more viral fusogenic proteins in a
pharmaceutically acceptable carrier.
[0038] In one embodiment, the composition comprises an expression
vector comprising a first expression cassette and a second
expression cassette, wherein the first expression cassette
comprises a first promoter sequence, one or more biologically
active RNA sequences directed to one or more target genes, a
recognition RNA sequence, optionally a terminal minihelix sequence,
a termination sequence, and optionally one or more primer
sequences, and the second expression cassette comprises a second
promoter sequence, an RNA binding domain sequence, a transport
peptide sequence, a poly A addition sequence, and optionally one or
more primer sequences and a pharmaceutically acceptable carrier. In
these embodiments, the biologically active RNA sequence(s), the
recognition RNA sequence, and the optional terminal minihelix
sequence are operably linked to the first promoter sequence and the
termination sequence and the RNA binding domain sequence and the
transport peptide sequence are operably linked to the second
promoter sequence and the poly A addition sequence.
[0039] In another embodiment, the composition comprises a first
expression vector comprising a first expression cassette, a second
expression cassette, and a third expression cassette, wherein the
first expression cassette comprises a first promoter sequence, one
or more biologically active RNA sequences directed to one or more
target genes, a recognition RNA sequence, optionally a terminal
minihelix sequence, a termination sequence, and optionally one or
more primer sequences, and the second expression cassette comprises
a second promoter sequence, an RNA binding domain sequence, a
transport peptide sequence, a poly A addition sequence, and
optionally one or more primer sequences, and the third expression
cassette comprises one or more promoter sequences, one or more
polynucleotide sequences encoding one or more viral polymerases and
one or more viral accessory proteins necessary for viral
replication, one or more polyA addition sequences, and optionally
one or more primers sequences, and a second expression vector
comprising a fourth expression cassette comprising one or more
promoter sequences, one or more polynucleotide sequences encoding
one or more viral coat proteins and one or more viral fusogenic
proteins, one or more polyA addition sequences, and optionally one
or more primers sequences, and a pharmaceutically acceptable
carrier. In these embodiments, the biologically active RNA
sequence(s), the recognition RNA sequence, and the optional
terminal minihelix sequence are operably linked to the first
promoter sequence and the termination sequence, the RNA binding
domain sequence and the transport peptide sequence are operably
linked to the second promoter sequence and the poly A addition
sequence, the polynucleotide sequence(s) encoding the viral
polymerase(s) and the viral accessory protein(s) is operably linked
to the one or more promoter sequences and the one or more polyA
addition sequences and the polynucleotide sequence(s) encoding the
viral coat proteins and the viral fusogenic proteins is operably
linked to the one or more promoter sequences and the one or more
polyA addition sequences.
[0040] The expression vectors and compositions of the invention can
be used to generate "bioreactor" cells which produce an RNA-protein
complex of the invention. The RNA portion of the RNA-protein
complex comprises one or more biologically active RNA sequences, a
recognition RNA sequence, and optionally a terminal minihelix
sequence. The protein portion of the RNA-complex comprises an RNA
binding domain and one or more transport peptide sequences. The
transcripts are exported from the cell nucleus to the cell
cytoplasm, where the transcript comprising the RNA binding domain
and the transport peptide sequence(s) is translated. The RNA
binding domain of the translated peptide interacts with the
recognition RNA sequence of the RNA portion, forming the
RNA-protein complex. The protein-RNA complex is subsequently
secreted from the cell and imported into the extracellular matrix
and/or neighboring cells where the biologically active RNA acts to
modulate gene expression.
[0041] In one embodiment, the invention provides a cell comprising
any of the expression vectors and compositions thereof provided
herein. In one embodiment, the invention provides a cell comprising
an expression vector comprising a polynucleotide sequence encoding
a nucleic acid comprising a biologically active RNA sequence, a
recognition RNA sequence, and optionally a terminal minihelix
sequence and a polynucleotide sequence encoding a polypeptide
comprising an RNA binding domain sequence and a transport
peptide.
[0042] In one embodiment, the invention provides a cell comprising
an expression vector comprising a polynucleotide sequence encoding
a nucleic acid comprising a biologically active RNA sequence, a
recognition RNA sequence, and optionally a terminal minihelix
sequence, a polynucleotide sequence encoding a polypeptide
comprising an RNA binding domain sequence and a transport peptide,
and one or more polynucleotide sequences encoding one or more viral
polymerases and one or more viral accessory proteins necessary for
viral replication and an expression vector comprising one or more
polynucleotide sequences encoding one or more viral coat proteins
and one or more viral fusogenic proteins.
[0043] In one embodiment, the invention provides a cell comprising
an expression vector comprising a polynucleotide sequence encoding
a nucleic acid comprising a biologically active RNA sequence, a
recognition RNA sequence, and optionally a terminal minihelix
sequence, a polynucleotide sequence encoding a polypeptide
comprising an RNA binding domain sequence and a transport peptide,
and an additional polynucleotide sequence encoding a nucleic acid
comprising one or more biologically active RNA sequences that
target one or more further gene target(s). In one embodiment, the
additional polynucleotide sequence encodes a nucleic acid
comprising one or more biologically active RNA sequences that
target a further gene target and an RNA recognition sequence. In
another embodiment, where one of the biologically active RNA
sequences in the vector is a short interfering RNA (siRNA),
double-stranded RNA (dsRNA), micro-RNA (miRNA), or short hairpin
RNA (shRNA), the additional polynucleotide sequence encodes a
nucleic acid comprising one or more biologically active RNA
sequences targeted to Dicer and/or Drosha.
[0044] In one embodiment, the invention provides a cell comprising
an expression vector comprising a polynucleotide sequence encoding
a nucleic acid comprising a biologically active RNA sequence, a
recognition RNA sequence, and optionally a terminal minihelix
sequence, a polynucleotide sequence encoding a polypeptide
comprising an RNA binding domain sequence and a transport peptide,
one or more polynucleotide sequences encoding one or more viral
polymerases and one or more viral accessory proteins necessary for
viral replication, and an additional polynucleotide sequence
encoding a nucleic acid comprising one or more biologically active
RNA sequences that target one or more further gene target(s) (for
example, Dicer and/or Drosha gene targets) and an expression vector
comprising one or more polynucleotide sequences encoding one or
more viral coat proteins and one or more viral fusogenic
proteins.
[0045] In one embodiment, the invention provides a cell comprising
an expression vector comprising a polynucleotide sequence encoding
a nucleic acid comprising a biologically active RNA sequence and
one or more polynucleotide sequences encoding one or more viral
polymerases and one or more viral accessory proteins necessary for
viral replication, and an expression vector comprising one or more
polynucleotide sequences encoding one or more viral coat proteins
and one or more viral fusogenic proteins.
[0046] In one embodiment, the invention provides a cell comprising
an expression vector comprising a polynucleotide sequence encoding
a nucleic acid comprising a biologically active RNA sequence, a
recognition RNA sequence, and optionally a terminal minihelix
sequence and an expression vector comprising a polynucleotide
sequence encoding a polypeptide comprising an RNA binding domain
sequence and one or more transport peptides. In one embodiment, the
cell further comprises a third expression vector comprising a
polynucleotide sequence encoding a nucleic acid comprising one or
more biologically active RNA sequences that target one or more gene
target(s) that differ from the gene target(s) of the biologically
active RNA in the first expression vector. In one embodiment, the
third expression vector comprises a polynucleotide sequence
encoding a nucleic acid comprising one or more biologically active
RNA sequences that target one or more gene targets and an RNA
recognition sequence. In another embodiment, where one of the
biologically active RNA sequences in the first expression vector is
a short interfering RNA (siRNA), double-stranded RNA (dsRNA),
micro-RNA (miRNA), or short hairpin RNA (shRNA), the third
expression vector comprises a polynucleotide sequence encoding a
nucleic acid comprising one or more biologically active RNA
sequences targeted to Dicer and/or Drosha.
[0047] The invention also provides a composition comprising a
bioreactor cell of the invention and a pharmaceutically acceptable
carrier. The composition can comprise any of the bioreactor cells
described herein and a pharmaceutically acceptable carrier. In one
embodiment, the composition comprises one or more cells comprising
an expression vector of the invention and a pharmaceutically
acceptable carrier. The cell can comprise one or more of any of the
expression vectors described herein. In one embodiment, the
invention provides a composition comprising one or more bioreactor
cells that express an RNA-complex of the invention and a
pharmaceutically acceptable carrier. In one embodiment, the
composition comprises one or more cells that express an RNA-protein
complex comprising one or more biologically active RNA sequences, a
recognition RNA sequence, optionally a terminal minihelix sequence,
an RNA binding domain, and one or more transport peptide sequences.
In one embodiment, the composition comprises one or more cells that
express an RNA-protein complex comprising one or more biologically
active RNA sequences, a recognition RNA sequence, optionally a
terminal minihelix sequence, an RNA binding domain, and a
cell-penetrating peptide sequence, and a pharmaceutically
acceptable carrier. In one embodiment, the composition comprises
one or more cells that express an RNA-protein complex comprising
one or more biologically active RNA sequences, a recognition RNA
sequence, optionally a terminal minihelix sequence, an RNA binding
domain, and a non-classical secretory domain and a pharmaceutically
acceptable carrier. In one embodiment, the composition comprises
one or more cells that express an RNA-protein complex comprising
one or more biologically active RNA sequences, a recognition RNA
sequence, optionally a terminal minihelix sequence, an RNA binding
domain, a cell-penetrating peptide sequence, and a non-classical
secretory domain and a pharmaceutically acceptable carrier.
[0048] Bioreactor cells comprising one or more expression vectors
of the invention are able to produce and secrete an RNA-protein
complex of the invention. The bioreactor cells are then useful in
vitro, ex vivo, and in vivo as novel transfection reagents for the
delivery of one or more biologically active RNA(s) to other target
cells and tissues. Accordingly, the invention provides a method for
producing a transfection reagent comprising one or more bioreactor
cells comprising the steps of: (a) preparing an expression vector
that encodes an RNA-protein complex comprising one or more
biologically active RNAs, a recognition RNA sequence, optionally a
terminal minihelix sequence, an RNA binding domain sequence, and
one or more transport peptide sequences (for example, selected from
a cell penetrating peptide, non-classical secretory domain,
endosomal release domain, receptor binding domain, and fusogenic
peptide sequence); (b) administering the expression vector of step
(a) to cells in culture to produce one or more bioreactor cells
expressing the RNA-protein complex; and (c) collecting the cultured
cells of step (b) as the transfection reagent. In one embodiment,
the method further comprises (d) testing the cells of (c) to
determine the bioreactor cells expressing the RNA-protein complex;
and (e) isolating the bioreactor cells from the other cells in
culture for use as the transfection reagent. The expression vector
can be any of the expression vectors described herein. The
RNA-protein complex can be any of the RNA-protein complexes
described herein. In one embodiment, the biologically active RNA of
the RNA-protein complex is an shRNA. In another embodiment, the
biologically active RNA of the RNA-protein complex is an aptamer.
In one embodiment, the cells of step (b) are stably transfected
with the expression vector.
[0049] In another embodiment, the invention provides a method for
producing a transfection reagent comprising one or more bioreactor
cells comprising the steps of: (a) preparing an expression vector
comprising a polynucleotide sequence that encodes a nucleic acid
comprising one or more biologically active RNAs, a recognition RNA
sequence, optionally a terminal minihelix sequence, a
polynucleotide sequence that encodes a polypeptide comprising an
RNA binding domain and one or more transport peptide sequences, and
an additional polynucleotide sequences that encodes a nucleic acid
comprising one or more biologically active RNA sequences that
target one or more further gene target(s); (b) administering the
expression vector of step (a) to cells in culture to produce one or
more bioreactor cells expressing the RNA-protein complex; and (c)
collecting the cultured cells of step (b) as the transfection
reagent. In one embodiment, the method further comprises (d)
testing the cells of (d) to determine the bioreactor cells
expressing the RNA-protein complex; and (e) isolating the
bioreactor cells from the other cells in culture for use as the
transfection reagent. In one embodiment, the additional
polynucleotide sequence encodes a nucleic acid comprising one or
more biologically active RNA sequences that target a further gene
target and an RNA recognition sequence. In another embodiment,
where one of the biologically active RNA sequences in the vector is
a short interfering RNA (siRNA), double-stranded RNA (dsRNA),
micro-RNA (miRNA), or short hairpin RNA (shRNA), the additional
polynucleotide sequence encodes a nucleic acid comprising one or
more biologically active RNA sequences targeted to Dicer and/or
Drosha.
[0050] In another embodiment, the invention provides a method for
producing a transfection reagent comprising one or more bioreactor
cells comprising the steps of: (a) preparing an expression vector
comprising a polynucleotide sequence that encodes a nucleic acid
comprising one or more biologically active RNAs, a recognition RNA
sequence, optionally a terminal minihelix sequence, a
polynucleotide sequence that encodes a polypeptide comprising an
RNA binding domain and one or more transport peptide sequences, and
one or more polynucleotide sequences encoding one or more viral
polymerases and one or more viral accessory proteins necessary for
viral replication; (b) preparing an expression vector comprising
one or more polynucleotide sequences encoding encoding one or more
viral coat proteins and one or more viral fusogenic proteins; (c)
administering the expression vector of step (a) and the expression
vector of step (b) to cells in culture to produce one or more
bioreactor cells (in this case, viral production cells) expressing
the RNA-protein complex; and (d) collecting the cultured cells of
step (c) as the transfection reagent. In one embodiment, the method
further comprises (e) testing the cells of (d) to determine the
bioreactor cells expressing the RNA-protein complex; and (f)
isolating the bioreactor cells from the other cells in culture for
use as the transfection reagent.
[0051] In another embodiment, the invention provides a method for
producing a transfection reagent comprising one or more bioreactor
cells comprising the steps of: (a) preparing an expression vector
comprising a polynucleotide sequence that encodes a nucleic acid
comprising one or more biologically active RNAs, a recognition RNA
sequence, optionally a terminal minihelix sequence, a
polynucleotide sequence that encodes a polypeptide comprising an
RNA binding domain and one or more transport peptide sequences, an
additional polynucleotide sequences that encodes a nucleic acid
comprising one or more biologically active RNA sequences that
target one or more further gene target(s), and one or more
polynucleotide sequences encoding one or more viral polymerases and
one or more viral accessory proteins necessary for viral
replication; (b) preparing an expression vector comprising one or
more polynucleotide sequences encoding encoding one or more viral
coat proteins and one or more viral fusogenic proteins; (c)
administering the expression vector of step (a) and the expression
vector of step (b) to cells in culture to produce one or more
bioreactor cells (in this case, viral production cells) expressing
the RNA-protein complex; and (d) collecting the cultured cells of
step (c) as the transfection reagent. In one embodiment, the method
further comprises (e) testing the cells of (d) to determine the
bioreactor cells expressing the RNA-protein complex; and (f)
isolating the bioreactor cells from the other cells in culture for
use as the transfection reagent. In one embodiment, the additional
polynucleotide sequence encodes a nucleic acid comprising one or
more biologically active RNA sequences that target a further gene
target and an RNA recognition sequence. In another embodiment,
where one of the biologically active RNA sequences in the vector is
a short interfering RNA (siRNA), double-stranded RNA (dsRNA),
micro-RNA (miRNA), or short hairpin RNA (shRNA), the additional
polynucleotide sequence encodes a nucleic acid comprising one or
more biologically active RNA sequences targeted to Dicer and/or
Drosha.
[0052] In another embodiment, the invention provides a method for
producing a transfection reagent comprising one or more bioreactor
cells comprising the steps of: (a) preparing an expression vector
comprising a polynucleotide sequence that encodes a nucleic acid
comprising one or more biologically active RNAs and one or more
polynucleotide sequences encoding one or more viral polymerases and
one or more viral accessory proteins necessary for viral
replication; (b) preparing an expression vector comprising one or
more polynucleotide sequences encoding encoding one or more viral
coat proteins and one or more viral fusogenic proteins; (c)
administering the expression vector of step (a) and the expression
vector of step (b) to cells in culture to produce one or more
bioreactor cells (in this case, viral production cells) expressing
the biologically active RNA; and (d) collecting the cultured cells
of step (c) as the transfection reagent. In one embodiment, the
method further comprises (e) testing the cells of (d) to determine
the bioreactor cells expressing the RNA-protein complex; and (f)
isolating the bioreactor cells from the other cells in culture for
use as the transfection reagent.
[0053] In another embodiment, the invention provides a method for
producing a transfection reagent comprising one or more bioreactor
cells comprising the steps of: (a) preparing an expression vector
comprising a polynucleotide sequence that encodes a nucleic acid
comprising one or more biologically active RNAs, a recognition RNA
sequence, and optionally a terminal minihelix sequence; (b)
preparing an expression vector comprising a polynucleotide sequence
that encodes a polypeptide comprising an RNA binding domain and one
or more transport peptide sequences; (c) administering the
expression vector of step (a) and the expression vector of step (b)
to cells in culture to produce one or more bioreactor cells
expressing the RNA-protein complex; and (d) collecting the cultured
cells of step (c) as the transfection reagent. In one embodiment,
the method further comprises (e) testing the cells of (d) to
determine the bioreactor cells expressing the RNA-protein complex;
and (f) isolating the bioreactor cells from the other cells in
culture for use as the transfection reagent.
[0054] The invention also provides methods of using the bioreactor
cells for the delivery of a biologically active RNA to target
cells, including target cells in vitro, ex vivo, and in vivo. In
one embodiment, the method of delivering a biologically active RNA
to target cells comprises the steps of: (a) preparing an expression
vector that encodes an RNA-protein complex comprising a
biologically active RNA, a recognition RNA sequence, optionally a
terminal minihelix sequence, an RNA binding domain, and one or more
transport peptide sequences selected from a cell penetrating
domain, non-classical secretory domain, endosomal release domain,
fusogenic peptide and a receptor binding domain; (b) administrating
the expression vector of step (a) to cells in culture to produce
bioreactor cells expressing the RNA-protein complex; (c) collecting
the cultured cells of step (b); and (d) mixing one or more target
cells with the cultured cell(s) collected in step (c) to deliver a
biologically active RNA to the target cells. In one embodiment, the
target cells are cells in culture. In another embodiment, the
target cells are cells in culture which have been obtained from a
subject, for example, a mammalian subject, including a human
subject. In one embodiment, the expression vector of step (a)
further comprises an additional polynucleotide sequences that
encodes a nucleic acid comprising one or more biologically active
RNA sequences that target one or more further gene target(s). In
one embodiment, the additional polynucleotide sequence encodes a
nucleic acid comprising one or more biologically active RNA
sequences that target a further gene target and an RNA recognition
sequence. In another embodiment, where one of the biologically
active RNA sequences in the vector is a short interfering RNA
(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), or short
hairpin RNA (shRNA), the additional polynucleotide sequence encodes
a nucleic acid comprising one or more biologically active RNA
sequences targeted to Dicer and/or Drosha.
[0055] In another embodiment, the method of delivering a
biologically active RNA to target cells comprises the steps of: (a)
preparing an expression vector comprising a polynucleotide sequence
that encodes a nucleic acid comprising one or more biologically
active RNAs, a recognition RNA sequence, optionally a terminal
minihelix sequence, a polynucleotide sequence that encodes a
polypeptide comprising an RNA binding domain and one or more
transport peptide sequences, and one or more polynucleotide
sequences encoding one or more viral polymerases and one or more
viral accessory proteins necessary for viral replication; (b)
preparing an expression vector comprising one or more
polynucleotide sequences encoding encoding one or more viral coat
proteins and one or more viral fusogenic proteins; (c)
administering the expression vector of step (a) and the expression
vector of step (b) to cells in culture to produce one or more
bioreactor cells (in this case, viral production cells) expressing
the RNA-protein complex; (d) collecting the cultured cells of step
(c); and (e) mixing one or more target cells with the cultured
cell(s) collected in step (d) to deliver a biologically active RNA
to the target cells. In one embodiment, the target cells are cells
in culture. In another embodiment, the target cells are cells in
culture which have been obtained from a subject, for example, a
mammalian subject, including a human subject. In one embodiment,
the expression vector of step (a) further comprises an additional
polynucleotide sequences that encodes a nucleic acid comprising one
or more biologically active RNA sequences that target one or more
further gene target(s). In one embodiment, the additional
polynucleotide sequence encodes a nucleic acid comprising one or
more biologically active RNA sequences that target a further gene
target and an RNA recognition sequence. In another embodiment,
where one of the biologically active RNA sequences in the vector is
a short interfering RNA (siRNA), double-stranded RNA (dsRNA),
micro-RNA (miRNA), or short hairpin RNA (shRNA), the additional
polynucleotide sequence encodes a nucleic acid comprising one or
more biologically active RNA sequences targeted to Dicer and/or
Drosha.
[0056] In another embodiment, the method for delivering a
biologically active RNA to target cells comprises the steps of: (a)
preparing an expression vector comprising a polynucleotide sequence
that encodes a nucleic acid comprising one or more biologically
active RNAs and one or more polynucleotide sequences encoding one
or more viral polymerases and one or more viral accessory proteins
necessary for viral replication; (b) preparing an expression vector
comprising one or more polynucleotide sequences encoding encoding
one or more viral coat proteins and one or more viral fusogenic
proteins; (c) administering the expression vector of step (a) and
the expression vector of step (b) to cells in culture to produce
one or more bioreactor cells (in this case, viral production cells)
expressing the biologically active RNA; (d) collecting the cultured
cells of step (c); and (e) mixing one or more target cells with the
cultured cell(s) collected in step (d) to deliver a biologically
active RNA to the target cells. In one embodiment, the target cells
are cells in culture. In another embodiment, the target cells are
cells in culture which have been obtained from a subject, for
example, a mammalian subject, including a human subject.
[0057] In another embodiment, the method for delivering a
biologically active RNA to target cells comprises the steps of: (a)
preparing an expression vector comprising a polynucleotide sequence
that encodes a nucleic acid comprising one or more biologically
active RNAs, a recognition RNA sequence, and optionally a terminal
minihelix sequence; (b) preparing an expression vector comprising a
polynucleotide sequence that encodes a polypeptide comprising an
RNA binding domain and one or more transport peptide sequences; (c)
administering the expression vector of step (a) and the expression
vector of step (b) to cells in culture to produce one or more
bioreactor cells expressing the RNA-protein complex; (d) collecting
the cultured cells of step (c); (e) mixing one or more target cells
with the cultured cell(s) collected in step (d) to deliver a
biologically active RNA to the target cells. In one embodiment, the
target cells are cells in culture. In another embodiment, the
target cells are cells in culture which have been obtained from a
subject, for example, a mammalian subject, including a human
subject.
[0058] In one embodiment, the target cells are cells which have
been removed from a subject, for example, a mammalian subject,
including a human subject. Thus, in one embodiment, the method of
delivering a biologically active RNA to target cells comprises the
steps of: (a) preparing an expression vector that encodes an
RNA-protein complex comprising a biologically active RNA, a
recognition RNA sequence, optionally a terminal minihelix sequence,
an RNA binding domain, and one or more transport peptide sequences
selected from a cell penetrating domain, non-classical secretory
domain, endosomal release domain, fusogenic peptide and a receptor
binding domain; (b) administrating the expression vector of step
(a) to cells in culture to produce bioreactor cells expressing the
RNA-protein complex; (c) collecting the cultured cells of step (b);
and (d) mixing one or more target cells removed from a subject with
the cultured cell(s) collected in step (c) to deliver a
biologically active RNA to the target cells. In one embodiment, the
method further comprises the step of administering the cells of
step (d) to a subject, for example, a mammalian subject, including
a human subject. In another embodiment, the method further
comprises the step of separating the bioreactor cells from the
target cells in step (d) before administering the target cells to
the subject. In one embodiment, the expression vector of step (a)
further comprises an additional polynucleotide sequences that
encodes a nucleic acid comprising one or more biologically active
RNA sequences that target one or more further gene target(s). In
one embodiment, the additional polynucleotide sequence encodes a
nucleic acid comprising one or more biologically active RNA
sequences that target a further gene target and an RNA recognition
sequence. In another embodiment, where one of the biologically
active RNA sequences in the vector is a short interfering RNA
(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), or short
hairpin RNA (shRNA), the additional polynucleotide sequence encodes
a nucleic acid comprising one or more biologically active RNA
sequences targeted to Dicer and/or Drosha.
[0059] In one embodiment, the method of delivering a biologically
active RNA to target cells comprises the steps of: (a) preparing an
expression vector comprising a polynucleotide sequence that encodes
a nucleic acid comprising one or more biologically active RNAs, a
recognition RNA sequence, optionally a terminal minihelix sequence,
a polynucleotide sequence that encodes a polypeptide comprising an
RNA binding domain and one or more transport peptide sequences, and
one or more polynucleotide sequences encoding one or more viral
polymerases and one or more viral accessory proteins necessary for
viral replication; (b) preparing an expression vector comprising
one or more polynucleotide sequences encoding encoding one or more
viral coat proteins and one or more viral fusogenic proteins; (c)
administering the expression vector of step (a) and the expression
vector of step (b) to cells in culture to produce one or more
bioreactor cells (in this case, viral production cells) expressing
the RNA-protein complex; (d) collecting the cultured cells of step
(c); and (e) mixing one or more target cells removed from a subject
with the cultured cell(s) collected in step (c) to deliver a
biologically active RNA to the target cells. In one embodiment, the
method further comprises the step of administering the cells of
step (e) to a subject, for example, a mammalian subject, including
a human subject. In another embodiment, the method further
comprises the step of separating the bioreactor cells from the
target cells in step (e) before administering the target cells to
the subject. In one embodiment, the expression vector of step (a)
further comprises an additional polynucleotide sequences that
encodes a nucleic acid comprising one or more biologically active
RNA sequences that target one or more further gene target(s). In
one embodiment, the additional polynucleotide sequence encodes a
nucleic acid comprising one or more biologically active RNA
sequences that target a further gene target and an RNA recognition
sequence. In another embodiment, where one of the biologically
active RNA sequences in the vector is a short interfering RNA
(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), or short
hairpin RNA (shRNA), the additional polynucleotide sequence encodes
a nucleic acid comprising one or more biologically active RNA
sequences targeted to Dicer and/or Drosha.
[0060] In another embodiment, the method for delivering a
biologically active RNA to target cells comprises the steps of: (a)
preparing an expression vector comprising a polynucleotide sequence
that encodes a nucleic acid comprising one or more biologically
active RNAs and one or more polynucleotide sequences encoding one
or more viral polymerases and one or more viral accessory proteins
necessary for viral replication; (b) preparing an expression vector
comprising one or more polynucleotide sequences encoding encoding
one or more viral coat proteins and one or more viral fusogenic
proteins; (c) administering the expression vector of step (a) and
the expression vector of step (b) to cells in culture to produce
one or more bioreactor cells (in this case, viral production cells)
expressing the biologically active RNA; (d) collecting the cultured
cells of step (c); and (e) mixing one or more target cells removed
from a subject with the cultured cell(s) collected in step (c) to
deliver a biologically active RNA to the target cells. In one
embodiment, the method further comprises the step of administering
the cells of step (e) to a subject, for example, a mammalian
subject, including a human subject. In another embodiment, the
method further comprises the step of separating the bioreactor
cells from the target cells in step (e) before administering the
target cells to the subject.
[0061] In another embodiment, the method for delivering a
biologically active RNA to target cells comprises the steps of: (a)
preparing an expression vector comprising a polynucleotide sequence
that encodes a nucleic acid comprising one or more biologically
active RNAs, a recognition RNA sequence, and optionally a terminal
minihelix sequence; (b) preparing an expression vector comprising a
polynucleotide sequence that encodes a polypeptide comprising an
RNA binding domain and one or more transport peptide sequences; (c)
administering the expression vector of step (a) and the expression
vector of step (b) to cells in culture to produce one or more
bioreactor cells expressing the RNA-protein complex; (d) collecting
the cultured cells of step (c); and (e) mixing one or more target
cells removed from a subject with the cultured cell(s) collected in
step (c) to deliver a biologically active RNA to the target cells.
In one embodiment, the method further comprises the step of
administering the cells of step (e) to a subject, for example, a
mammalian subject, including a human subject. In another
embodiment, the method further comprises the step of separating the
bioreactor cells from the target cells in step (e) before
administering the target cells to the subject.
[0062] The invention provides methods for secreting one or more
biologically active RNA molecules from a bioreactor cell and
methods for modulating target gene expression in vivo, ex vivo, and
in vitro. The invention provides an expression vector designed to
produce an RNA-protein complex comprising at least one biologically
active RNA molecule targeting one or more genes of interest and a
fusion protein capable of delivering the biologically active RNA
molecule(s) to the extracellular matrix and/or neighboring cells
and tissues. The administration of the expression vector to cells
in vivo, ex vivo, and in vitro converts the cells into
"bioreactors" that produce and deliver biologically active RNA
molecules, secreted as RNA-protein complexes, to the extracellular
matrix and/or other neighboring cells. Thus, the RNA-mediated
effect is amplified through the production and delivery of
biologically active RNAs to surrounding cells and tissues.
[0063] In one embodiment, the invention provides a method for
modulating the expression of one or more target gene(s) in a
subject comprising administering to the subject one or more
expression vectors of the invention. In another embodiment, the
invention provides a method for modulating the expression of one or
more target gene(s) in a subject comprising administering to the
subject a composition comprising one or more expression vectors of
the invention and a pharmaceutically acceptable carrier. In another
embodiment, the invention provides a method for modulating the
expression of one or more target gene(s) in a subject comprising
administering to the subject a cell comprising one or more
expression vectors of the invention and a pharmaceutically
acceptable carrier. The expression vector can be any of the
expression vectors of the invention described herein.
[0064] In one embodiment, the invention provides a method for
modulating the expression of one or more target gene(s) in a
subject comprising administering to the subject one or more
bioreactor cells of the invention. In another embodiment, the
invention provides a method for modulating the expression of one or
more target gene(s) in a subject comprising administering to the
subject a composition comprising one or more bioreactor cells of
the invention and a pharmaceutically acceptable carrier, including
but not limited to phosphate buffered saline (PBS), saline, or 5%
dextrose. The bioreactor cell(s) can be any of the bioreactor
cells(s) of the invention described herein. In one embodiment, the
bioreactor cell(s) produces and secretes an RNA-protein complex
comprising one or more biologically active RNA sequences directed
to a target gene(s), a recognition RNA sequence, optionally a
terminal minihelix sequence, an RNA binding domain sequence, and
one or more transport peptide sequences, for example, selected from
a cell penetrating peptide sequence, non-classical secretory
domain, endosomal release domain, receptor binding domain, and
fusogenic peptide.
[0065] In any of the methods of modulating gene expression in a
subject described herein, the subject can be a mammalian subject,
including, for example, a human, rodent, murine, bovine, canine,
feline, sheep, equine, and simian subject.
[0066] The invention additionally provides a method of preventing,
ameliorating, and/or treating a disease or condition associated
with defective gene expression and/or activity in a subject
comprising administering to the subject one or more expression
vectors of the invention. In one embodiment, the invention provides
a method of preventing, ameliorating, and/or treating a disease or
condition associated with defective gene expression and/or activity
in a subject comprising administering to the subject a composition
comprising one or more expression vectors of the invention and a
pharmaceutically acceptable carrier. In one embodiment, the
invention provides a method of preventing, ameliorating, and/or
treating a disease or condition associated with defective gene
expression and/or activity in a subject comprising administering to
the subject a cell comprising one or more expression vectors of the
invention and a pharmaceutically acceptable carrier. The expression
vector can be any of the expression vectors of the invention
described herein.
[0067] In one specific embodiment, the invention provides a method
for modulating the expression of a target gene in a target cell
comprising administering to the target cell an expression vector of
the invention, wherein the target cell produces and secretes an
RNA-protein complex of the invention and wherein the RNA-protein
complex is subsequently delivered to the extracellular matrix or to
other target cells. In another embodiment, the invention provides a
method for modulating the expression of a target gene in a target
cell comprising administering to the target cell a composition
comprising an expression vector of the invention, wherein the
target cell produces and secretes an RNA-protein complex of the
invention and wherein the RNA-protein complex is subsequently
delivered to the extracellular matrix or to other target cells. In
another embodiment, the invention provides a method for modulating
the expression of a target gene in a target cell comprising
administering to the target cell a cell comprising an expression
vector of the invention, wherein the target cell produces and
secretes an RNA-protein complex of the invention and wherein the
RNA-protein complex is subsequently delivered to the extracellular
matrix or to other target cells. The expression vector can be any
expression vector of the invention described herein.
[0068] The invention also provides methods for modulating the
expression of a target gene in a target cell ex vivo. In one
embodiment, the invention provides a method for modulating the
expression of a target gene in a target cell ex vivo comprising
administering to the target cell ex vivo one or more expression
vectors of the invention. In another embodiment, the invention
provides a method for modulating the expression of a target gene in
a target cell ex vivo comprising administering to the target cell
ex vivo a composition comprising one or more expression vectors of
the invention and a pharmaceutically acceptable carrier. In another
embodiment, the invention provides a method for modulating the
expression of a target gene in a target cell ex vivo comprising
administering to the target cell ex vivo a bioreactor cell
comprising one or more expression vectors of the invention and a
pharmaceutically acceptable carrier. The expression vector can be
any of the expression vectors of the invention described
herein.
[0069] The invention also provides methods for modulating gene
expression in a cell in culture. In one embodiment, the invention
provides a method for modulating the expression of one or more
target gene(s) in a cell in culture comprising administering to the
cell one or more expression vectors of the invention. In another
embodiment, the invention provides a method for modulating the
expression of one or more target gene(s) in a cell in culture
comprising administering to the cell a composition comprising one
or more expression vectors of the invention and a pharmaceutically
acceptable carrier. In another embodiment, the invention provides a
method for modulating the expression of one or more target gene(s)
in a cell in culture comprising administering to the cell a a
bioreactor cells comprising one or more expression vectors of the
invention and a pharmaceutically acceptable carrier. The expression
vector can be any of the expression vectors of the invention
described herein.
[0070] In one embodiment, the invention provides a method for
modulating the expression of one or more target gene(s) in a cell
in culture comprising administering to the cell a first expression
vector encoding a nucleic acid comprising one or more biologically
active RNA sequences directed to a target gene, a recognition RNA
sequence, and optionally a terminal minihelix sequence and a second
expression vector encoding a polypeptide comprising an RNA binding
domain and one or more transport peptide sequences, for example,
selected from a cell penetrating peptide sequence, non-classical
secretory domain, endosomal release domain, and a receptor binding
domain.
[0071] In addition the present invention provides expression
vectors constructed from a replication defective or replication
incompetent viral particles which carry and distribute one or more
biologically active RNA molecules from a transformed packaging
cell. In one embodiment, the invention provides a viral vector
comprising a polynucleotide that encodes any of the nucleic acid
molecules described herein. In one embodiment, the invention
provides a viral vector comprising a polynucleotide that encodes a
nucleic acid molecule comprising one or more biologically active
RNA sequences and a recognition RNA sequence. In another
embodiment, the invention provides a viral vector comprising a
polynucleotide that encodes a nucleic acid molecule comprising one
or more biologically active RNA sequences, a recognition RNA
sequence, and a terminal minihelix sequence. The biologically
active RNA sequence can be any of the biologically active RNA
sequences described herein and otherwise known in the art. In one
embodiment, the viral vector comprises a polynucleotide encoding a
nucleic acid molecule wherein the biologically active RNA sequence
is selected from a ribozyme, antisense nucleic acid, allozyme,
aptamer, short interfering RNA (siRNA), double-stranded RNA
(dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), and a
transcript encoding one or more biologically active peptides. In
one specific embodiment, the viral vector comprises a
polynucleotide encoding a nucleic acid molecule wherein the
biologically active RNA sequence is a short hairpin RNA (shRNA). In
one specific embodiment, the viral vector comprises a
polynucleotide encoding a nucleic acid molecule wherein the
biologically active RNA sequence is an aptamer. The recognition RNA
sequence can be any of the recognition RNA sequences described
herein and otherwise known in the art. In one embodiment, viral
vector vector comprises a polynucleotide encoding a nucleic acid
molecule wherein the recognition RNA sequence is selected from a U1
loop, Group II intron, NRE stem loop, S1A stem loop, Bacteriophage
BoxBR, HTV Rev response element, AMVCP recognition sequence, and
ARE sequence. The terminal minihelix sequence can be any of the
terminal minihelix sequences described herein and otherwise known
in the art. In one embodiment, the terminal minihelix sequence is
selected from the adenovirus VA1 RNA molecule.
[0072] In another embodiment, the viral vector additionally
comprises a polynucleotide that encodes a nucleic acid molecule
comprising one or more biologically active RNA sequences targeted
to Dicer and/or Drosha. None of these polynucleotides encode an RNA
binding domain. In one embodiment, the polynucleotide encodes a
nucleic acid molecule comprising a single biologically active RNA
sequence. In another embodiment, the polynucleotide encodes a
nucleic acid molecule comprising two or more biologically active
RNA sequences. In certain embodiments, the biologically active RNA
sequence is selected from a ribozyme, antisense nucleic acid,
allozyme, aptamer, short interfering RNA (siRNA), double-stranded
RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), and a
transcript encoding one or more biologically active peptides.
[0073] In any of the above-described embodiments of the viral
vector comprising a polynucleotide encoding a nucleic acid molecule
of the invention, the polynucleotide can comprise a sequence
wherein the recognition RNA sequence, the individual biologically
active RNA sequences, the optional terminal minihelix sequence, and
any other included sequences are joined with the addition of one or
more intervening or additional sequences or are joined directly
without the addition of intervening sequences.
[0074] In another embodiment, the viral vector comprises a
polynucleotide encoding a polypeptide comprising an RNA binding
domain, and one or more transport peptide sequences selected from a
cell penetrating peptide, a non-classical secretory domain, a
receptor binding domain, an endosomal release domain, and a
fusogenic peptide. In one embodiment, the polynucleotide encoding
the polypeptide further comprises a promoter sequence, a
termination sequence, and optionally one or more primers sequences.
In another embodiment, the viral vector additionally comprises a
polynucleotide that encodes a nucleic acid molecule comprising one
or more biologically active RNA sequences, a recognition RNA
sequence, and optionally a terminal minihelix sequence. In yet a
further embodiment the polynucleotide encoding the nucleic acid
molecule additionally comprises a promoter sequence, a termination
sequence, and optionally one or more primer sequences. In yet
another embodiment, the viral vector additionally comprises a
polynucleotide that encodes a nucleic acid molecule comprising one
or more biologically active RNA sequences targeted to Dicer and/or
Drosha, and optionally a promoter sequence, a termination sequence,
and one or more primer sequences. Thus, in one embodiment, the
viral vector comprises a polynucleotide encoding a polypeptide
comprising an RNA binding domain, and one or more transport
peptides selected from a cell penetrating peptide, a non-classical
secretory domain, a receptor binding domain, an endosomal release
domain, and a fusogenic peptide, and further comprises a
polynucleotide that encodes a nucleic acid molecule comprising one
or more biologically active RNA sequences, a recognition RNA
sequence, optionally a terminal minihelix sequence. In one
embodiment, this viral vector can further comprise a polynucleotide
that encodes a nucleic acid molecule comprising one or more
biologically active RNA sequences targeted to Dicer and/or Drosha.
In any of these embodiments, the viral vector can optionally
comprise one or more promoter sequences, one or more termination
sequences, and one or more primer sequences.
[0075] In any of the above-described embodiments of the viral
vector, the polynucleotide can comprise a sequence wherein any of
the RNA binding domain, cell penetrating peptide, non-classical
secretory domain, receptor binding domain, endosomal release
domain, fusogenic peptide, and any other included sequences (i.e.,
promoter, termination, primer, biologically active RNA, recognition
RNA, terminal minihelix sequences, etc.) are joined with the
addition of one or more intervening or additional sequences or are
joined directly without the addition of intervening sequences. In
any of the above-described embodiments, the vector can comprise a
polynucleotide that encodes a polypeptide wherein the sequence or
sequences of the individual domains and peptides are joined without
or with the addition of one or more linker, spacer, or other
sequences.
[0076] The present invention also provides engineered, replication
defective virus to deliver biologically active RNAs from
transformed packaging cells to target cells. In one embodiment the
invention provides packaging cells generated by transfection of
recipient cells with plasmids encoding for the two independent
viral RNAs, one encoding the virus structural genes, the other
encoding the non-structural genes and a biologically active RNA
sequence. In one embodiment the viral non-structural and structural
genes are selected from DNA viruses and RNA viruses with
non-limiting examples of suitable viruses being Adenovirus,
Adeno-Associated Virus, Herpes Simplex Virus Lentivirus,
Retrovirus, Sindbis virus, Foamy virus. The biologically active RNA
sequence can be any of the biologically active RNA sequences
described herein and otherwise known in the art. In one embodiment,
the biologically active RNA sequence is selected from a ribozyme,
antisense nucleic acid, allozyme, aptamer, short interfering RNA
(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short
hairpin RNA (shRNA), and a transcript encoding one or more
biologically active peptides. In one specific embodiment, the
biologically active RNA sequence is a short hairpin RNA (shRNA). In
another specific embodiment, the biologically active RNA sequence
is micro-RNA (miRNA).
[0077] Successful co-transfection of both plasmids yield packaging
cells capable of producing replication defective viral particles.
In one embodiment the invention provides packaging cells produced
by transfection of cells in vitro, ex vivo or in vivo. In a further
embodiment packaging cells are collected and mixed with target
cells in vitro. In another embodiment packaging cells are collected
and administered in target cells in vivo. In a further embodiment
packaging cells are collected and transferred to target cell ex
vivo.
BRIEF DESCRIPTION OF THE DRAWINGS
[0078] FIG. 1 is a non-limiting schematic exemplifying the in vivo
mechanism of action for the vector-based delivery of a biologically
active RNA molecule, which exemplary biologically active RNA
molecule is a shRNA. As shown, the expression vector (pBioR)
expresses a nucleic acid molecule comprising a recognition RNA
sequence and an shRNA and a fusion protein comprising an RNA
binding domain (RBD) and a cell penetrating peptide (CPP). The
fusion protein is translated in the cytoplasm where the RNA binding
domain of the translated fusion protein binds to the recognition
RNA sequence of the nucleic acid, forming an RNA-protein complex.
The RNA-protein complex is secreted into the extracellular matrix
and taken up by neighboring cells where the shRNA acts to modulate
the target gene of interest (GOI).
[0079] FIG. 2 is a non-limiting schematic exemplifying the in vivo
mechanism of action for the vector-based delivery of a biologically
active RNA molecule, which exemplary biologically active RNA
molecule is a shRNA. As shown, the expression vector (pBioR)
expresses a nucleic acid molecule comprising a recognition RNA
sequence and an shRNA and a fusion protein comprising an RNA
binding domain (RBD), a non-classical secretory domain (NCS), and a
cell penetrating peptide (CPP). The fusion protein is translated in
the cytoplasm where the RNA binding domain of the translated fusion
protein binds to the recognition RNA sequence of the nucleic acid,
forming an RNA-protein complex. The RNA-protein complex is secreted
into the extracellular matrix and taken up by neighboring cells
where the shRNA acts to modulate the target gene of interest
(GOI).
[0080] FIG. 3 is a non-limiting schematic exemplifying the in vivo
mechanism of action for the vector-based delivery of a biologically
active RNA molecule, which exemplary biologically active RNA
molecule is an aptamer targeting a specific cell-surface receptor.
As shown, the expression vector (pBioR) expresses a nucleic acid
molecule comprising a recognition RNA sequence and an aptamer
targeting a specific cell-surface receptor and a fusion protein
comprising an RNA binding domain (RBD) and a non-classical
secretory domain (NCS). The fusion protein is translated in the
cytoplasm where the RNA binding domain of the translated fusion
protein binds to the recognition RNA sequence of the nucleic acid,
forming an RNA-protein complex. The RNA-protein complex is secreted
into the extracellular matrix. The aptamer binds to the target
cell-surface receptor, preventing the receptor ligand from binding
the receptor.
[0081] FIG. 4 is a non-limiting schematic exemplifying the in vivo
mechanism of action for the vector-based delivery of a biologically
active RNA molecule, which exemplary biologically active RNA
molecule is an aptamer targeting a specific extracellular matrix
protein. As shown, the expression vector (pBioR) expresses a
nucleic acid molecule comprising a recognition RNA sequence and an
aptamer targeting a specific extracellular matrix protein and a
fusion protein comprising an RNA binding domain (RBD) and a
non-classical secretory domain (NCS). The fusion protein is
translated in the cytoplasm where the RNA binding domain of the
translated fusion protein binds to the recognition RNA sequence of
the nucleic acid, forming an RNA-protein complex. The RNA-protein
complex is secreted into the extracellular matrix. The aptamer
binds to the extracellular matrix protein, preventing the
extracellular matrix protein from entering a target cell. The
extracellular matrix protein can be, among other things, a
cell-surface receptor ligand, whereby the aptamer binds the ligand
and prevents it from binding to its receptor (not shown).
[0082] FIG. 5 shows a schematic diagram of the backbone plasmid
pEGEN 1.1. pEGEN 1.1 includes an SV40 promoter sequence (1), an
intronic sequence (2), a multiple cloning sequence (MCS), a human
growth hormone poly-A tail sequence (4), a kanamycin resistance
gene (7) and a pUC origin of replication (8).
[0083] FIG. 6 shows a schematic diagram of the backbone plasmid
pEGEN 2.1. pEGEN 2.1 includes a chicken-actin promoter sequence
(1), an intronic sequence (2), a multiple cloning sequence (MCS), a
human growth hormone poly-A tail sequence (4), a kanamycin
resistance gene (7) and a pUC origin of replication (8).
[0084] FIG. 7 shows a schematic diagram of the backbone plasmid
pEGEN 3.1. pEGEN 3.1 includes a CMV promoter sequence (1), an
intronic sequence (2), a multiple cloning sequence (MCS), a human
growth hormone poly-A tail sequence (4), a kanamycin resistance
gene (7) and a pUC origin of replication (8).
[0085] FIG. 8 shows a schematic diagram of the backbone plasmid
pEGEN 4.1. pEGEN 4.1 includes a human U6 promoter sequence (1), a
multiple cloning sequence (MCS), a polyT terminator sequence (4), a
kanamycin resistance gene (7) and a pUC origin of replication
(8).
[0086] FIG. 9 shows a schematic diagram of the expression vector
pBioR Pol II which encodes an exemplary RNA-protein complex of the
invention. The vector includes an SV40 promoter (1) and an intronic
sequence (2) upstream of an Sec-RNA sequence (3) and a downstream
hGH polyA sequence (4). The vector also comprises a .beta.-actin
promoter (5) upstream of a fusion protein sequence (6) and a
downstream hGH polyA sequence (4). The vector also comprises a
kanamycin resistance gene (7) and a pUC origin of replication
(8).
[0087] FIG. 10 shows a schematic diagram of expression vector pBioR
Pol III which encodes an exemplary RNA-protein complex of the
invention. The vector includes an hU6 promoter upstream (1) and an
intronic sequence (2) upstream of an Sec-RNA sequence (3) and a
downstream Pol-III poly-T terminator sequence (4). The vector also
comprises a .beta.-actin promoter (5) upstream of a fusion protein
sequence (6) and a downstream hGH polyA sequence (4). The vector
also comprises a kanamycin resistance gene (7) and a pUC origin of
replication (8).
[0088] FIG. 11 shows a schematic diagram of expression vector pBioR
Pol II combo which encodes an exemplary RNA-protein complex of the
invention. The vector includes a .beta.-actin promoter (1), an
intronic sequence (2), a fusion protein cassette (6), a Sec-RNA
cassette (3) with flanking introns (2) internal to the fusion
protein, a human growth hormone poly-A tail sequence (4), a
kanamycin resistance gene (7) and a pUC origin of replication
(8).
[0089] FIG. 12 shows a schematic diagram of expression vector pBioR
Pol II stable which encodes an exemplary RNA-protein complex of the
invention. The vector includes a CTS regulator (9), a PGK promoter
(1), a puromycin resistance gene (10), a chicken-actin promoter
(5), a fusion protein cassette (6), a Sec-RNA cassette (3) with
flanking introns (2) internal to the fusion protein, a human growth
hormone poly-A tail sequence (4), a kanamycin resistance gene (7)
and a pUC origin of replication (8).
[0090] FIG. 13 shows a schematic diagram of expression vector pBioR
Pol II Dicer which encodes an exemplary RNA-protein complex of the
invention. The vector includes a SV40 promoter (1), an intronic
sequence (2), an shRNA sequence (3), a hGH poly-A tail sequence
(4), a chicken .beta.-actin promoter (5), a fusion protein cassette
(6), a Sec-RNA cassette (11) with flanking introns (2) internal to
the fusion protein, a human growth hormone poly-A tail sequence
(4), a kanamycin resistance gene (7) and a pUC origin of
replication (8).
[0091] FIG. 14A is a non-limiting schematic showing an exemplary
transfection assay to generate bioreactor cells and test their
secretory activity using the CPP-Luciferase/CPP-Alkaline
Phosphatase reporter system. FIG. 14B presents results for TAT
mediated secretion of the luciferase reporter protein from CT26
cells. CT26 cells were transfected with plasmids expressing
luciferase or a CPP-Luciferase fusion protein. CPP domains assayed
include TAT, REV, FHV, and Penetratin (Pen). After 48 hours, cell
media was replaced with PBS and cells were incubated at 37.degree.
C. for an additional 1 hour, 3 hours, or 6 hours. The PBS
supernatant was collected and the cells were lysed in TENT buffer.
Luciferase activity was measured for equivalent amounts of
solubilized cellular protein and PBS supernatant using standard
methods. The relative luciferase activity present in cellular and
supernatant fractions is presented as a percentage of the total
luciferase activity observed in both fractions.
[0092] FIGS. 15A and 15B show schematic diagrams for the
construction of plasmids for expression of secreted RNAs and
bioreactor fusion proteins. As shown in FIG. 15A, pE3.1
Sec-Reporter includes a CMV promoter sequence (1), an intronic
sequence (2), a secreted RNA reporter coding sequence (Box B
sequence and glucagon-like peptide 1) (3), a human growth hormone
poly-A tail sequence (4), a kanamycin resistance gene (7) and a pUC
origin of replication (8). As shown in FIG. 15B, pE1 TAT-RBD
includes an SV40 promoter sequence (1), an intronic sequence (2), a
fusion protein coding sequence (i.e., an RNA binding domain (RBD)
and cell penetrating peptide (TAT)) (6), a human growth hormone
poly-A tail sequence (4), a kanamycin resistance gene (7) and a pUC
origin of replication (8). FIGS. 15C-E show the restriction enzyme
analyses of the pE3.1 Sec-Reporter and pE1 TAT-RBD plasmids. FIG.
15C shows the restriction enzyme analysis of the pE3.1
Sec-Reporter, in which a novel EcoNI restriction site is introduced
with the RNA expressing insert. FIGS. 15D and 15E show the
restriction enzyme and PCR analyses, respectfully, of two pE1
TAT-RBD plasmids: one expressing a fusion protein with the TAT cell
penetrating peptide fused to a Protein N RNA binding domain (TAT+),
the other expressing a fusion protein with the TAT cell penetrating
peptide fused to a Rev RNA binding domain (TAT-). In these figures,
(M) denotes a size marker lane. In FIG. 15C, Sec-Reporter (-)
refers to the pE3.1 Sec-Reporter plasmid only and Sec-Reporter (+)
refers to the pE3.1 Sec-Reporter plasmid with the RNA expressing
insert. In FIGS. 15D and 15E, p1.1 refers to the pE1.1 plasmid
only, TAT(-) refers to the pE1.1 plasmid with the fusion protein
insert comprising a TAT cell penetrating peptide fused to a Rev RNA
binding domain, and TAT(+) refers to the pE1.1 plasmid with the
fusion protein insert comprising a TAT cell penetrating peptide
fused to a Protein N RNA binding domain.
[0093] FIGS. 16A and 16B show the expression products for the
secreted RNAs and the bioreactor fusion proteins. For the secreted
RNA reporter transcript analyses shown in FIG. 16A, CT26 cells were
transfected with pE3.1 Sec-Reporter (FIG. 15A). After 48 hours,
total cellular RNA was collected from untreated control cells and
transfected cells, and purified RNA was amplified using RT-PCR and
separated on 3% low melt agarose gels (1.times.TAE). Untransfected
control cells ("U") show only the 18S rRNA internal control (18S)
whereas the transfected cells show both the 18S rRNA product and
the parent reporter RNA product ("R"), which corresponds to the
plasmid only, or the secreted reporter RNA product ("SR"), which
corresponds to the plasmid and the Sec-RNA sequence insert. FIG.
16B shows the fusion protein expression analyses, in which CT26
cells were transfected with plasmids expressing the bioreactor
fusion protein. After 48 hours, cell lysates from untreated cells
and cells transfected with pE3.1 Sec-Reporter and either pE1.1
TAT+(TAT fused to a Protein N RNA binding domain and 6.times.
Histidine epitope tag) or pE2.1TAT+(TAT fused to a Protein N RNA
binding domain and 6.times. Histidine epitope tag) were spotted to
PVDF membranes along with a positive control protein for the
blotting antibody. The blots were developed with chromogenic
substrates and recorded with an image documentation center. "His+"
shows the results of the positive control and "Unt" shows the
results of untransfected CT26 cells. The blots were developed with
chromogenic substrates and recorded with an image documentation
center. "His+" shows the chromogenic signal obtained with a
purified His-tagged protein (positive control); "Unt" shows the
background signal obtained with protein lysates collected from
untransfected CHO cells; pE1.1 TAT+ shows the signal obtained with
protein lysates collected from CHO cells transfected with pE1.1
TAT-Protein N-6.times.His; and pE2.1 TAT+ shows the signal obtained
with protein lysates collected from CHO cells transfected with
pE2.1 TAT-Protein N-6.times.His.
[0094] FIGS. 17A and 17B show bioreactor activity using the two
component plasmids described in FIGS. 15A and 15B. RNA from
untreated control CT26 cells and CT26 cells transfected with the
pE3.1 Sec-Reporter and pE1TAT-RBD plasmids expressing the secreted
RNAs and the bioreactor fusion proteins was collected and used as
template for RT-PCR amplification reactions. RNA was also collected
from the cell culture media, purified and amplified. The amplified
products were separated on 3% low melt agarose gels (1.times.TAE)
along with DNA size standards. FIGS. 17A and 17B show the results
of a transfection assay with pE3.1 Sec-Reporter and either pE1.1
TAT(+) (TAT fused to the proper RBD) or pE1.1 TAT(-) (TAT fused to
a negative control RBD). The left hand panel of FIG. 17A shows
RT-PCR products for cell lysates collected from cells transfected
with the parent reporter plasmid ("R"), the reporter plasmid
containing the sec-RNA sequence insert ("SR"), the sec-RNA reporter
plasmid co-transfected with pE1.1 TAT(+) ("TAT(+)"; TAT fused to a
Protein N RNA binding domain) or with pE1.1 TAT(-) ("TAT(-)"; TAT
fused to a Rev RNA binding domain, serving as a negative control
RBD). The right hand panel of FIG. 17A shows both cell lysates
("C") and extracellular media samples ("M") from cells
cotransfected with the sec-RNA reporter plasmid and pE1.1 TAT(+)
("TAT(+)"; TAT fused to a Protein N RNA binding domain) or pE1.1
TAT(-) ("TAT(-)"; fused to a Rev RNA binding domain). FIG. 17B
shows the results of a second assay, identical to the first, where
steps have been taken to eliminate the 18S rRNA contamination of
the media observed in the first experiment.
[0095] FIG. 18 is a non-limiting schematic showing an exemplary
transfection assay to generate and test the import activity of
bioreactor cells using the GFP reporter system.
[0096] FIG. 19A is a schematic showing the secretion and activity
of aptamers targeted to Oncostatin M produced by bioreactor cells
of the invention. FIG. 19B is a non-limiting schematic showing an
exemplary transfection assay to determine the secretion activity of
bioreactor cells using a reporter system and a secreted RNA aptamer
targeting the Oncostatin M protein, an activator of the gp130
receptor mediated signaling pathway.
[0097] FIG. 20A is a schematic showing the secretion and activity
of aptamers targeted to HER3 produced by bioreactor cells of the
invention. FIG. 20B is a non-limiting schematic showing an
exemplary transfection assay to determine the secretion activity of
bioreactor cells using a reporter system and a secreted RNA aptamer
targeting the HER3.
[0098] FIG. 21 is a non-limiting schematic showing an exemplary
transfection assay to determine the secretion activity of
bioreactor cells and subsequent delivery of an inhibitory shRNA to
the cytoplasm of a target cell.
[0099] FIG. 22 is a non-limiting schematic showing the two
constructs required for producing the viral packaging cells
containing a biologically active inhibitory RNA molecule.
[0100] FIG. 23 is a non-limiting schematic showing the production
of viral packaging cells containing virus particles and a
biologically active RNA molecule. The schematic further exemplifies
the transfer of the biologically active RNA molecule into a target
cell.
[0101] FIG. 24 is a non-limiting schematic showing the production
of viral packaging cells containing virus particles, the bioreactor
fusion protein and a biologically active RNA molecule. The
schematic further exemplifies the transfer of the bioreactor
expression cassettes via the virus particle to primary target cells
(secondary bioreactor cells) and subsequent transfer of the
biologically active RNA molecule into secondary target cells.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0102] As used herein the term "biologically active RNA" is meant
to refer to any RNA sequence that modulates gene expression or gene
activity of targeted gene products.
[0103] As used herein, the term "recognition RNA sequence" is meant
to refer to any RNA sequence that is specifically bound by a
peptide comprising an RNA binding domain.
[0104] As used herein, the term "RNA binding domain" is meant to
refer to any protein or peptide sequence that specifically binds to
a corresponding recognition RNA sequence.
[0105] As used herein, the term "transport peptide" is meant to
refer to any peptide sequence that facilitates movement of any
attached cargo within a cell or cells, including facilitating cargo
movement across a cell membrane of a cell, secretion of cargo from
a cell, and release of cargo from an endosome, as well as other
means of cellular movement. In specific, but non-limiting examples,
the transport peptide can be a sequence derived from a cell
penetrating peptide, a non-classical secretory sequence, an
endosomal release domain, a receptor binding domain, and a
fusogenic peptide.
[0106] As used herein, the term "cell penetrating peptide" is meant
to refer to any peptide sequence that facilitates movement of any
attached cargo across a lipid bilayer, such as the membrane of a
cell.
[0107] As used herein, the term "non-classical secretory sequence"
is meant to refer to any protein or peptide sequence that provides
for secretion of any attached cargo from a cell via an ER-Golgi
independent pathway.
[0108] As used herein, the term "endosomal release domain" is meant
to refer to any peptide sequence that facilitates release of any
attached cargo from the endosome of a cell.
[0109] As used herein, the term "receptor binding domain" is meant
to refer to any RNA or protein domain capable of interacting with a
surface bound cellular receptor.
[0110] As used herein, the term "fusogenic peptide" is meant to
refer to any peptide sequence that facilitates cargo exit from the
endosome of a cell.
[0111] As used herein, the term "sec-RNA" refers to the RNA portion
of the RNA-protein complex of the invention. Typically, the
"sec-RNA" comprises one or more biologically active RNAs, a
recognition RNA sequence, and optionally a terminal minihelix
sequence. When complexed with a fusion protein of the invention,
the sec-RNA is secreted from the cell.
[0112] As used herein, the term "sec-shRNA" refers to the shRNA
portion of the RNA-protein complex of the invention. Typically, the
"sec-shRNA" comprises one or more short hairpin RNAs, a recognition
RNA sequence, and optionally a terminal minihelix sequence. When
complexed with a fusion protein of the invention, the sec-shRNA is
secreted from the cell.
[0113] As used herein, the term "fusion protein" is meant to refer
to at least two polypeptides, typically from different sources,
which are operably linked. With regard to polypeptides, the term
operably linked is intended to mean that the two polypeptides are
connected in a manner such that each polypeptide can serve its
intended function. Typically, the two polypeptides are covalently
attached through peptide bonds. The fusion protein can be produced
by standard recombinant DNA techniques. For example, a DNA molecule
encoding the first polypeptide is ligated to another DNA molecule
encoding the second polypeptide, and the resultant hybrid DNA
molecule is expressed in a host cell to produce the fusion protein.
The DNA molecules are ligated to each other in a 5' to 3'
orientation such that, after ligation, the translational frame of
the encoded polypeptides is not altered (i.e., the DNA molecules
are ligated to each other in-frame). In a specific example, a
fusion protein refers to a peptide comprising an RNA binding domain
sequence and one or more transport peptide sequences.
[0114] As used herein, the term "bioreactor cell" or "bioreactor"
is meant to refer to any cell that produces and secretes a Sec-RNA
molecule.
[0115] As used herein, the term "pBioR plasmid" is meant to refer
to any plasmid comprising a polynucleotide encoding at least an RNA
binding domain sequence, a transport peptide sequence, and a
polynucleotide encoding a biologically active RNA and a recognition
RNA sequence.
[0116] As used herein, the term "expression cassette" is meant to
refer to a nucleic acid sequence capable of directing expression of
a particular nucleotide sequence, which may include a promoter
operably linked to a nucleotide sequence of interest that may be
operably linked to termination signals. It also may include
sequences required for proper translation of the nucleotide
sequence. The coding region can code for a peptide of interest but
may also code for a biologically active RNA of interest. The
expression cassette including the nucleotide sequence of interest
may be chimeric. The expression cassette may also be one that is
naturally occurring but has been obtained in a recombinant form
useful for heterologous expression. In a specific example, an
expression cassette comprises a nucleic acid sequence comprising a
promoter sequence, a polynucleotide encoding a peptide sequence or
a polynucleotide encoding an RNA sequence, and a terminator
sequence.
[0117] The term "operatively linked" is used herein to refer to an
arrangement of flanking sequences wherein the flanking sequences so
described are configured or assembled so as to perform their usual
function. A flanking sequence operably linked to a coding sequence
may be capable of effecting the replication, transcription and/or
translation of the coding sequence. For example, a coding sequence
is operably linked to a promoter when the promoter is capable of
directing transcription of that coding sequence. A flanking
sequence need not be contiguous with the coding sequence, so long
as it functions correctly. Thus, for example, intervening
untranslated yet transcribed sequences can be present between a
promoter sequence and the coding sequence and the promoter sequence
can still be considered "operably linked" to the coding
sequence.
[0118] Mechanism of Action for the Vector Based Delivery System
[0119] The invention provides a vector based RNA delivery system in
which a plasmid converts a transfected cell into an RNA bioreactor
capable of producing and secreting biologically active RNA
molecules. The plasmid accomplishes this by encoding both the
biologically active RNA molecule and a fusion protein capable of
facilitating its secretion from the bioreactor and delivery to the
extracellular matrix and/or surrounding target cells. Once
delivered to the target cells, the biologically active RNA molecule
functions as it would in any cell. This approach directly addresses
the key issue in application of plasmid based RNAi mediated
therapeutics, namely the low transfection efficiencies associated
with plasmid delivery. Although the initial transfection of the
bioreactor cells may be limited do to the technical difficulties
associated with standard gene delivery methods, the subsequent
expression of the plasmid based delivery system of the present
invention will mitigate the traditional limitations as they permit
sustained and continued delivery of active RNAs and associated
proteins from bioreactor cells. RNA-mediated knockdown is amplified
through bioreactor cell cellular production and delivery of
biologically active RNAs to surrounding cells and tissues.
[0120] The central component of the plasmid based delivery system
is the fusion protein that facilitates secretion and/or delivery.
Classical export of protein molecules through the ER-Golgi is
co-translational, meaning the proteins are translocated across the
ER membrane as they are being made. This prevents the use of
classical transport mechanisms in the bioreactor cell, as the RNA
binding domain would only briefly exist in the cytoplasm with the
Sec-RNA molecule and transport across the membrane would likely
disrupt the RNA-protein interaction. Instead, a fully translated
and folded protein in the cytoplasm can be subsequently secreted
via a non-classical mechanism with the biologically active RNA
cargo in tow. A growing number of proteins are now known to be
secreted via non-classical pathways which are independent of the
ER-Golgi apparatus. Although the precise mechanism of export for
these systems is not fully characterized, the proteins are known to
be translated in the cytoplasm and therefore contain sequence
motifs that allow them to be secreted and are suitable for use in
the bioreactor.
[0121] An early step in bioreactor cell function is synthesis of
the RNA and protein components of the RNA-protein complex and
localization of those components to the cell cytoplasm. Promoter
driven transcription of the RNA molecules occurs via well
established mechanisms and can be optimized for the cell type being
used as the bioreactor. Export of the transcript encoding the
fusion protein follows typical Pol-II mRNA pathway via the nuclear
pore complex. Alternatively, the Sec-RNA molecule can be
constructed in such a way that it is exported via the exportin-5
pathway utilized by microRNAs and shRNAs. Still alternatively, the
RNA molecule can contain an adenovirus VA1 minihelix domain to
facilitate export of the Sec-RNA from the nucleus. It is also
possible to express the Sec-RNA construct from a Pol-II promoter
and terminate with an hGH poly-adenylation signal, such that the
Sec-RNA can be capped and exported from the nucleus via the nuclear
pore complex.
[0122] Once co-localized in the cytoplasm, the biologically active
RNA and fusion protein must come together to form the RNA-protein
complex. This binding event involves a specific, high affinity
interaction that provides a homogenous population of stable
complexes which is achieved by including a high affinity RNA
binding domain in the fusion protein and a corresponding sequence
specific recognition site in the nucleic acid comprising the
biologically active RNA molecule. The RNA binding domain and the
RNA recognition sequence interact in the cytoplasm of the
bioreactor cell and couple the biologically active RNA sequence to
the protein machinery required for secretion and delivery to target
cells. The specificity of the interaction minimizes the secretion
of other RNAs endogenous to the bioreactor cell and the high
affinity helps maintain the complexes in the extracellular
space.
[0123] RNA-Protein Complexes
[0124] As discussed, the invention provides a vector based RNA
delivery system in which a plasmid converts a transfected cell into
an RNA bioreactor capable of producing and secreting biologically
active RNA molecules. The bioreactor plasmid has the capacity to
encode and distribute any biologically active RNA molecule linked
to the recognition sequence for the delivery fusion protein. Thus,
the expression vectors of the invention comprise polynucleotide
sequences encoding nucleic acid comprising one or more biologically
active RNA sequences, an RNA recognition sequence, and optionally a
terminal mini-helix sequence and/or polynucleotide sequences
encoding a polypeptide comprising an RNA binding domain and one or
more transport peptide sequences. The biologically active RNA
molecules can exert a biological effect through a number of
different mechanisms depending on the cellular components with
which they interact. Most of the biologically active RNAs function
through base pairing interactions with specific mRNA transcripts
that lead to translational silencing or degradation of the mRNA
molecule. Two related classes of inhibitory RNAs are antisense RNA
molecules and small inhibitory RNA molecules. The antisense RNA is
typically a direct complement of the mRNA transcript it targets and
functions by presenting an obstacle to the translational machinery
and also by targeting the transcript for degradation by cellular
nucleases. The small inhibitory RNA (siRNA) molecules act through
the post-transcriptional gene silencing (PTGS) pathway or through
the RNA interference (RNAi) pathway. These RNAs are about 22
nucleotides in length and associate with specific cellular proteins
to form RNA-induced silencing complexes (RISCs). These small RNAs
are also complementary to sequences within their mRNA targets and
binding of these complexes leads to translational silencing or
degradation of the transcripts.
[0125] Two additional classes of RNA molecules that can modulate
gene expression are the catalytic RNA ribozymes and the RNA
aptamers. Ribozymes are RNA based enzymes that catalyze chemical
reactions on RNA substrates, most often hydrolysis of the
phosphodiester backbone. Formation of the catalytic active site
requires base pairing between the ribozyme and the RNA substrate,
so ribozyme activity can also be targeted to desired substrates by
providing appropriate guide sequences. When targeted to mRNA
transcripts, ribozymes have the potential to degrade those
transcripts and lead to downregulation of the associated protein.
RNA aptamers are typically selected from pools of random RNA
sequences by their ability to interact with a target molecule,
often a protein molecule. Engineering RNA aptamers is less
straightforward as the binding is not defined by base pairing
interactions, but once an effective sequence is found the
specificity and affinity of the binding often rivals that of
antibody-antigen interactions. RNA aptamers also have a greater
range of target molecules and the potential to alter gene activity
via a number of different mechanisms. This includes direct
inhibition of the biological activity of the target molecule with
no requirement for degradation of the protein or the mRNA
transcript which produces it.
[0126] In certain embodiments of the invention, the one or more
biologically active RNA sequences of the RNA-protein complex is
selected from a ribozyme, antisense nucleic acid, allozyme,
aptamer, short interfering RNA (siRNA), double-stranded RNA
(dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), and a
transcript encoding one or more biologically active peptides, and
any combination thereof. In one embodiment, one or more of the
biologically active RNA sequences is a short hairpin RNA (shRNA).
In another embodiment, one or more of the biologically active RNA
sequences is an aptamer. With respect to biologically active RNAs
that are a transcript encoding one or more biologically active
peptides, exemplary peptides include those selected from a peptide
encoded by a tumor suppressor gene, a pro-apoptotic factor, and an
intrabody for a cancer system or a protein that restores gene
function in a disease system resulting from loss of function or
deletion mutations. The biologically active RNA sequence of the
nucleic acid molecule can be directed to any target gene of
interest. For example, the biologically active RNA can be directed
to any gene found in any publicly available gene sequence database,
including, for example, any of the databases found in the National
Center for Biotech Information (NCBI). In one specific embodiment,
the biologically active RNA sequence is a short hairpin RNA
(shRNA). In another specific embodiment, the biologically active
RNA is an aptamer. Non-limiting examples of suitable shRNA
sequences include Mmp2, Vascular Endothelial Growth Factor (VEGF),
Vascular Endothelial Growth Factor Receptor (VEGFR), Caveolin-1
(Cav-1), Epidermal Growth Factor Receptor (EGFR),
Harvey--retrovirus associated DNA sequences (H-Ras), B-cell
CCL/lymphoma 2 (Bcl-2), Survivin, Focal adhesion kinase (FAK),
Signal transducer and activator of transcription 3 (STAT-3), Human
epidermal growth-factor receptor 3 (HER-3), Beta-Catenin, and Src
shRNA sequences, among others described herein and known in the
art. Table I provides the nucleotide sequences of non-limiting
exemplary biologically active RNA sequences. In certain
embodiments, the biologically active RNA sequence comprises one or
more sequences selected from any of SEQ ID NOs: 1-15.
[0127] The nucleic acid comprising a biologically active RNA
sequence additionally comprises a recognition RNA sequence, which
sequence is recognized by and specifically binds to an RNA binding
domain located in a fusion protein of the invention. Numerous
examples of specific, high affinity interactions between
recognition RNA sequences (in RNA sequences) and RNA binding
domains (in protein sequences) are known and described in the art.
The recognition RNA sequence of the invention can be any RNA
sequence described in the art known to bind an RNA binding domain
of a polypeptide. In one embodiment, the recognition RNA sequence
is at least about 10 nucleotides in length. In one embodiment, the
recognition RNA sequence is from about 10 nucleotides to about 250
nucleotides. In certain specific embodiments, the recognition RNA
sequence is, for example, about 10-15 nucleotides, about 16-20
nucleotides, about 21-25 nucleotides, about 26-30 nucleotides,
about 31-35 nucleotides, about 36-40 nucleotides, about 41-45
nucleotides, about 46-50 nucleotides, about 51-75 nucleotides,
about 76-100 nucleotides, about 101-125 nucleotides, about 126-150
nucleotides, about 151-175 nucleotides, about 176-200 nucleotides,
or about 201-250 nucleotides. In one embodiment, the recognition
RNA sequence has a dissociation constant (K.sub.d) of at least
about 100 nM. In a specific embodiment, the dissociation constant
is from about 100 nM to about 1 pM, Non-limiting examples of
specific, high affinity interactions between recognition RNA
sequences (in RNA sequences) and RNA binding domains (in protein
sequences) include U1 loop sequence with U1A sequence, Domain I or
Domain IV of Group II intron sequence with CRS 1 sequence, NRE stem
loop sequence with nucleolin sequence, S1A stem loop sequence with
hRBMY sequence, Bacteriophage BoxBR sequence with Bacteriophage
Protein N, HIV Rev response element with HIV Rev protein, alfalfa
mosaic virus coat protein recognition sequence (AMVCP) with AMVCP
protein, and ARE stem loop sequence with tristetrapolin sequence,
among others. In certain specific embodiments, the recognition RNA
sequence of the nucleic acid comprises a sequence selected from a
U1 loop, Group II intron, NRE stem loop, S1A stem loop,
Bacteriophage BoxBR, HIV Rev response element, alfalfa mosaic virus
coat protein recognition sequence (AMVCP), and ARE sequence. Table
II provides the nucleotide sequences of non-limiting exemplary
recognition RNA sequences. In certain specific embodiments, the
recognition RNA sequence comprises the sequence of any of SEQ ID
NOs: 16-23.
[0128] In certain embodiments, the nucleic acid molecule comprises
one or more biologically active RNA sequences, a recognition RNA
sequence, and a terminal minihelix sequence. Terminal minihelix
sequences are short sequences of about 17 nucleotides that anneal
the 5' and 3' ends of the RNA molecule. This sequence has been
shown to facilitate nuclear export of RNA molecules derived from
Pol-III promoters and may help drive formation of the RNA-fusion
protein complexes in the BioReactor cells. Examples of suitable
terminal minihelix sequences are described herein and otherwise
known in the art. In one embodiment, the terminal minihelix
sequence is at least about 17 nucleotides in length. In a specific
embodiment, the terminal minihelix sequence is from about 10
nucleotides to about 100 nucleotides in length. In one embodiment,
the terminal minihelix sequence is from the adenovirus VA1 RNA
molecule.
[0129] In addition, the expression vectors of the invention can
comprise one or more polynucleotide sequences encoding polypeptides
comprising one or more biologically active RNA sequences targeted
to Dicer and/or Drosha. None of the sequences of these embodiments
contain an RNA recognition sequence. Such polypeptides are useful
when one or more of the biologically active RNA sequences is a
short interfering RNA (siRNA), double-stranded RNA (dsRNA),
micro-RNA (miRNA), and short hairpin RNA (shRNA).
[0130] In any of the above-described nucleic acid molecules, the
nucleic acid molecule can comprise a sequence wherein the
recognition RNA sequence, the individual biologically active RNA
sequences, and the optional terminal minihelix sequence are joined
directly without the addition of intervening or additional
sequences. Alternatively, in any of the above-described nucleic
acid molecules, the nucleic acid molecule can comprise a sequence
wherein one or more of the sequences comprising the recognition RNA
sequence, the individual biologically active RNA sequences, and the
optional terminal minihelix sequence are joined with the addition
of one or more intervening or additional sequences. Likewise, in
any of the above-described nucleic acid molecules, the nucleic acid
molecule can comprise a sequence wherein the individual
biologically active RNA sequences themselves are joined directly
without the addition of one or more intervening or additional
sequences or are joined with the addition of one or more
intervening or additional sequences.
[0131] The ability of the BioReactor cell to secrete and deliver
biologically active RNA molecules to neighboring cells derives from
the properties of the RNA-protein complex produced from the pBioR
plasmid or plasmids. First, the fusion proteins (comprising an RNA
binding domain and optionally other sequences) bind to the
biologically active RNAs (via the RNA recognition sequence) and are
secreted from the bioreactor cell. In the extracellular matrix, the
RNA-protein complex remains intact long enough to reach the target
cells. Once at the surface of the target cell, the fusion protein
facilitates import of the biologically active RNA to the cytoplasm
of the target cell.
[0132] The secretion of the RNA-protein complex is optimized by
efficient binding of the Sec-RNA by the RNA binding domain of the
fusion protein. To drive formation of fusion protein-Sec-RNA
complexes, the fusion proteins contain high affinity RNA binding
domains of viral or bacterial origin. The utilization of non-native
high affinity interaction improves the chances of obtaining a
homogenous population of stable complexes with minimal competition
from non-specific binding of RNA molecules endogenous to the
bioreactor cell.
[0133] Thus, in one embodiment, the fusion protein comprises an RNA
binding domain and one or more transport peptides. The RNA binding
domain of the novel fusion protein can be any amino acid sequence
capable of recognizing a corresponding RNA recognition motif. In
one embodiment, the RNA binding domain is from about 25 amino acids
to about 300 amino acids. In certain specific embodiments, the RNA
binding domain is, for example, about 25-49 amino acids, about
50-75 amino acids, about 76-100 amino acids, about 101-125 amino
acids, about 126-150 amino acids, about 151-175 amino acids, about
176-200 amino acids, about 201-225 amino acids, about 226-250 amino
acids, about 251-275 amino acids, or about 276-300 amino acids. The
RNA binding domain of the fusion polypeptide can be any RNA binding
domain known and described in the art. In certain specific
embodiments, the RNA binding domain of the fusion polypeptide
comprises an amino acid sequence selected from a U1A, CRS1, CRM1,
Nucleolin RBD12, hRBMY, Bacteriophage Protein N, HIV Rev, alfalfa
mosaic virus coat protein (AMVCP), and tristetrapolin amino acid
sequence. The amino acid sequences of non-limiting examples of RNA
binding domain sequences are shown in Table III. In certain
specific embodiments, the RNA binding domain comprises a sequence
selected from any of SEQ ID NOs: 24-31.
[0134] Another component of the fusion protein is the domain that
facilitates secretion of the RNA-protein complex. Proteins that
follow the non-classical secretory pathway lack the typical
secretory signal that directs the classical export mechanism, are
excluded from the ER-Golgi network and can be secreted in the
presence of drugs that inhibit ER-Golgi transport. Several
mechanisms have been proposed for the non-classical secretion
pathway, including membrane blebbing, vesicular and non-vesicular
non-classical transport, active and passive membrane transporters
and membrane flip-flop. Peptide sequences from proteins that access
secretion pathways independent from those of the ER-Golgi network
are useful in the secretion of the biologically active RNA
molecules of the invention. Another group of sequences useful for
facilitating secretion of the RNA-protein complexes are the cell
penetrating peptides. The precise mechanism of entry for these
peptides is not fully known, but may involve the endosomal pathway,
although some data suggests non-endosomal mechanisms.
[0135] The transport peptide of the fusion polypeptide can be any
amino acid sequence that facilitates the delivery of nucleic acids,
peptides, fusion proteins, RNA-protein complexes, and/or other
biological molecules to the extracellular matrix and/or to
neighboring cells and tissues. One example of a transport peptide
is a cell penetrating peptide which facilitates import of the
Sec-RNA into the target cell. There are numerous cell penetrating
peptides known in the art which peptide sequences are able to cross
the plasma membrane. Such peptides are often present in
transcription factors, such as the homeodomain proteins and viral
proteins, such as TAT of HIV-1. Delivery of RNA-protein complexes
to the cytoplasm of cells via cell penetrating peptides has been
established experimentally. For example, delivery of siRNAs to CHO
cells with a purified fusion protein consisting of the U1A RNA
binding domain and the TAT cell penetrating peptide has been
reported. Additional reports utilizing a biotin-streptavidin
linkage also show successful delivery of various cargo molecules
via the TAT peptide. Although TAT mediated delivery of cargo
molecules to the cytoplasm of target cells does not appear to
require an additional fusogenic peptide to facilitate endosomal
release, the addition of such a peptide to TAT can improve the
efficiency of delivery. The necessity of fusogenic peptides as part
of the delivery system may depend on the identity of the cell
penetrating peptide used in the fusion protein.
[0136] Thus, in one embodiment, the transport protein is a cell
penetrating peptide. Typically such sequences are polycationic or
amphiphilic sequences rich in amino acids with positively charged
side groups, i.e., basic amino acids such as histidine, lysine, and
arginine. Numerous examples of cell penetrating peptides are known
and described in the art. Non-limiting examples of suitable cell
penetrating peptides include those derived from protein membrane
transduction domains which are present in transcription factors,
such as the homeodomain proteins, and viral proteins, such as TAT
of HIV-1. In one embodiment, the cell penetrating peptide is from
about 10 amino acids to about 50 amino acids, including for
example, about 10-15 amino acids, about 16-20 amino acids, about
21-25 amino acids, about 26-30 amino acids, about 31-35 amino
acids, about 36-40 amino acids, about 41-45 amino acids, and about
46-50 amino acids. In certain specific embodiments, the cell
penetrating peptide of the polypeptide comprises an amino acid
sequence selected from a penetratin, transportan, MAP, HIV TAT,
Antp, Rev, FHV coat protein, TP10, and pVEC amino acid sequence.
The amino acid sequences of non-limiting examples of cell
penetrating peptide sequences are shown in Table IV. In certain
specific embodiments, the cell penetrating peptide comprises a
sequence selected from any of SEQ ID NOs: 32-40.
[0137] Another example of a transport peptide is a non-classical
secretory domain. The non-classical secretory domain can be any
amino acid sequence that directs a peptide and/or other biological
molecule to be secreted from a cell via a pathway other than the
classical pathway(s) of protein secretion. The biological molecule
can be secreted into the extracellular matrix and/or can be
delivered to surrounding cells and tissues. Numerous examples of
non-classical secretory domains are known and described in the art.
In one embodiment, the non-classical secretory domain is from about
50 amino acids to about 250 amino acids. In certain specific
embodiments, the non-classical secretory domain is, for example,
about 50-75 amino acids, about 76-100 amino acids, about 101-125
amino acids, about 126-150 amino acids, about 151-175 amino acids,
about 176-200 amino acids, about 201-225 amino acids, or about
226-250 amino acids. In certain specific embodiments, the
non-classical secretory domain comprises an amino acid sequence
selected from Galcetin-1 peptide, Galectin-3 peptide, IL-1.alpha.,
IL-1.beta., HASPB, HMGB1, FGF-1, FGF-2, IL-2 signal, secretory
transglutaminase, annexin-1, HIV TAT, Herpes VP22, thioredoxin,
Rhodanese, and plasminogen activator signal amino acid sequences.
Non-limiting examples of non-classical secretory domain sequences
are shown in Table V. In certain specific embodiments, the
non-classical secretory domain comprises a sequence selected from
any of SEQ ID NOs: 41-48.
[0138] Other examples of suitable transport peptides include, but
are not limited to sequences derived from a receptor binding
domain, a fusogenic peptide, and an endosomal release domain. In
one embodiment, the transport peptide comprises a sequence derived
from a receptor binding domain. The receptor binding domain can be
any amino acid sequence that specifically binds to a surface
receptor complex on the extracellular side the target cell
membrane. In certain specific embodiments, the receptor binding
domain comprises an amino acid sequence selected from the EGF
protein, the VEGF protein, the vascular homing peptide, the gp30
protein (or other Erb B-2 binding protein), or the galectin-1
protein (or other CA125 binding protein).
[0139] In another embodiment, the transport peptide comprises a
sequence derived from an endosomal release domain. The endosomal
release domain can be any amino acid sequence that facilitates
release of the RNA-protein complex from the endosomal compartment
of the target cell. In certain specific embodiments, the endosomal
release domain comprises an amino acid sequence selected from the
Hemagglutanin protein from influenza, the E1 protein from Semliki
Forrest Virus, or a polyhistidine motif.
[0140] In another embodiment, the transport peptide comprises a
sequence derived from fusogenic peptide. Table VI provides
non-limiting examples of suitable fusogenic peptides. Thus, in
certain specific embodiments, the fusogenic peptide comprises a
sequence selected from any of SEQ ID NOs: 50-54.
[0141] In any of the above-described embodiments of the fusion
protein polypeptide, the polypeptide can comprise a sequence or
sequences wherein the individual domains and peptides are joined
directly without the addition of one or more linker, spacer, or
other sequences. In another embodiment, the polypeptide can
comprise a sequence or sequences wherein the individual domains and
peptides are joined with the addition of one or more linker,
spacer, and/or other sequences.
[0142] Thus, in certain specific embodiments of the expression
vectors of the invention, the biologically active RNA sequence(s)
is selected from a ribozyme, antisense nucleic acid, allozyme,
aptamer, short interfering RNA (siRNA), double-stranded RNA
(dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), and a
transcript encoding one or more biologically active peptides. The
recognition RNA sequence is selected from a U1 loop, Group II
intron, NRE stem loop, S1A stem loop, Bacteriophage BoxBR, HIV Rev
response element, AMVCP recognition sequence, and ARE sequence. The
RNA binding domain comprises an amino acid sequence derived from a
U1A, CRS1, CRM1, Nucleolin RBD12, hRBMY, Bacteriopage Protein N,
HIV Rev, AMVCP, and tristetrapolin amino acid sequence. The
transport peptide is selected from a cell penetrating peptide, a
non-classical secretory domain, a receptor binding domain, a
fusogenic peptide, and an endosomal release domain. Suitable cell
penetrating peptide sequences include, but are not limited to,
those peptides having amino acid sequences derived from a
penetratin, transportan, MAP, HIV TAT, Antp, Rev, FHV coat protein,
TP10, and pVEC amino acid sequence. Suitable non-classical
secretory domain sequences include, but are not limited to,
peptides having amino acid sequence derived from Galcetin-1
peptide, Galectin-3 peptide, IL-1.alpha., IL-1.beta., HASPB, HMGB1,
FGF-1, FGF-2, IL-2 signal, secretory transglutaminase, annexin-1,
HIV TAT, Herpes VP22, thioredoxin, Rhodanese, and plasminogen
activator signal amino acid sequences. Suitable fusogenic peptide
sequences include, but are not limited to, peptides having amino
acid sequence derived from HA from influenza, Gp41 from HIV,
Melittin, GALA, and KALA.
[0143] In any of the embodiments described herein of the
RNA-protein complex, the nucleic acid molecule can comprise a
sequence wherein the recognition RNA sequence, the individual
biologically active RNA sequences, and the optional terminal
minihelix sequence are joined directly without the addition of one
or more intervening or additional sequences. Alternatively, in any
of the above-described embodiments of the RNA-protein complex, the
nucleic acid molecule can comprise a sequence wherein the
recognition RNA sequence, the individual biologically active RNA
sequences, and the optional terminal minihelix sequence are joined
with the addition of one or more intervening or additional
sequences. In any of the above-described embodiments of the
RNA-protein complex, the nucleic acid molecule can comprise a
sequence wherein the individual biologically active RNA sequences
themselves are joined with or without the addition of one or more
intervening or additional sequences. In any of the above-described
embodiments of the RNA-protein complex, the polypeptide portion of
the RNA-protein complex can comprise a sequence or sequences
wherein any of the individual domains and peptides are joined with
or without the addition of linker, spacer, and/or other
sequences.
[0144] Expression Vectors
[0145] In one embodiment, the expression vector comprises a first
expression cassette comprising polynucleotide sequence that encodes
an RNA molecule comprising one or more biologically active RNA
sequences, a recognition RNA site for an RNA binding domain
(Sec-RNA), and optionally a mini-terminal helix sequence. The
expression vector further comprises a second expression cassette
comprising polynucleotide sequence that encodes a fusion protein
comprising an RNA binding domain and one or more transport peptides
that facilitate secretion of the RNA-protein complex and delivery
of the biologically active RNA to the extracellular matrix or to
target cells. In a further embodiment, the expression vector
additionally comprises a third expression cassette, wherein the
third expression cassette comprises one or more polynucleotide
sequences encoding one or more viral polymerases and one or more
viral accessory proteins necessary for viral replication.
Optionally, the expression vector can additionally comprise a
fourth expression cassette, or, a separate expression vector can
comprise an expression cassette, which comprises polynucleotide
sequence encoding one or more biologically active RNAs, optionally
a recognition RNA sequence, and optionally a terminal minihelix
sequence. In one embodiment, the one or more biologically active
RNA sequences of the fourth expression cassette are directed to a
target gene, which may or may not be the same target gene targeted
by the biologically active RNA sequence(s) of the first expression
cassette. In another embodiment, the one or more biologically
active RNA sequences of the fourth expression cassette are directed
to the Dicer protein and/or the Drosha protein within the
bioreactor cell. This cassette does not contain a recognition RNA
sequence for the RNA binding domain and therefore is not secreted
from the bioreactor cell.
[0146] In one embodiment, the first and second expression cassettes
are combined by placing the Sec-RNA sequence into artificial
introns within the RNA encoding the fusion protein. This vector
offers the advantages of reducing the overall plasmid size and
places the transcription of all BioReactor components under the
control of a single promoter. Upon administration of the expression
vector to a cell, the RNA-protein complex can be expressed from the
vector as a single RNA transcript or as one or more RNA
transcripts. For example, the RNA-protein complex can be expressed
from the vector as a single transcript comprising the RNA portion
of the RNA-protein complex (comprising one or more biologically
active RNA sequences, a recognition RNA sequence, and optionally a
terminal minihelix sequence) and the protein portion of the
RNA-complex (comprising an RNA binding domain and one or more
transport peptide sequences selected from, for example, a
cell-penetrating peptide, a non-classical secretory domain, an
endosomal release domain, fusogenic peptide and a receptor binding
domain). The Sec-RNA is encoded within an artificial intron placed
in either the 5' untranslated region (UTR) or within the coding
sequence for the fusion protein. The Sec-RNA sequence is subcloned
between the splice donor and splice acceptor sites of the
artificial intron using appropriate restriction sites. After
transcription, the Sec-RNA is released from the mRNA encoding the
fusion protein by the splicing machinery endogenous to the
bioreactor cell. The separate transcripts are exported from the
cell nucleus to the cell cytoplasm, where the transcript comprising
the RNA binding domain sequence and optional other sequence(s) are
translated. The RNA binding domain of the translated peptide
interacts with the recognition RNA sequence of the RNA, forming the
RNA-protein complex.
[0147] In other embodiments, the first and second expression
cassettes, and optional third and fourth expression cassettes
additionally comprise one or more sequences selected from a
promoter sequence, a sequence comprising on or more restriction
enzyme sites, a primer sequence, GC base pair sequence, initiation
codon, translational start site, and a termination sequence.
Suitable promoters include Pol II promoters, including but not
limited to, Simian Virus 40 (SV40), Cytomegalovirus (CMV),
.beta.-actin, human albumin, human HIF-.alpha., human gelsolin,
human CA-125, ubiquitin, and PSA promoters. In another embodiment,
the promoter is a Pol III promoter. Non-limiting examples of
suitable Pol III promoters include, but are not limited to, human
H1 and human U6 promoters. In another embodiment, the cassette
additionally comprises one or more termination sequences.
Non-limiting examples of suitable termination sequences include,
but are not limited to, a human growth hormone (hGH)
polyadenylation sequence, a bovine growth hormone (BGH)
polyadenylation sequence, a Simian Virus 40 (SV40) large T
polyadenylation sequence, and a Herpes Simplex Virus Thymidine
Kinase (HSV-tk) polyadenylation sequence. In one embodiment, the
expression cassettes additionally comprises one or more primer
sequences, which may contain restriction enzyme sites, one or more
promoter sequences, and one or more termination sequences.
[0148] In any of the above-described embodiments of the expression
vector, the polynucleotide can comprise sequence wherein any of the
biologically active RNA sequences, recognition RNA sequence, RNA
binding domain sequence, transport peptide sequence, viral
polypeptides, and any other included sequences (i.e., promoter,
termination sequence, primer, etc.) are joined with the addition of
one or more intervening or additional sequences or are joined
directly without the addition of intervening sequences. In any of
the above-described embodiments, the expression vector can comprise
a polynucleotide that encodes a polypeptide wherein the sequence or
sequences of the individual domains and peptides are joined without
or with the addition of one or more linker, spacer, or other
sequences.
[0149] In a further embodiments, the expression vector additionally
comprises one or more multiple cloning site sequences. Also, the
expression vector can additionally comprise one or more drug
resistance gene sequences. Examples of suitable drug resistant
genes include, but are not limited to, kanamycin, ampicillin,
puromycin, tetracycline, and chloramphenicol resistant genes, as
well as any other drug resistant genes known and described in the
art. The expression vector can additionally comprise a pUC origin
of replication.
[0150] Expression cassettes for the protein or RNA components of
the bioreactor plasmid are prepared by PCR amplification of the
relevant sequences from cDNA clones or RNA expressing plasmids,
respectively, using the appropriate forward and reverse primers.
Primers include sequences complementary to the domain of interest
or biologically active RNA sequence, sites for restriction enzymes
used in subcloning and about six GC base pairs at the 5' end of
each primer to facilitate digestion with restriction enzymes. The
recognition RNA sequence is added to the primer corresponding to
the 5' end of the biologically active RNA sequence in order to
generate the Sec-RNA expression construct. This expression
construct is digested with appropriate restriction enzymes for
subcloning into the pEGEN4.1 construct, which places the Sec-RNA
expression cassette downstream from a human U6 promoter sequence
and upstream of a Pol III poly-T termination sequence.
Alternatively, the Sec-RNA expression cassette can be subcloned
into pEGEN3.1, which places RNA expression under the control of the
CMV Pol-II promoter and terminates with a human GH poly-adenylation
signal.
[0151] Several exemplary expression vectors are shown in FIGS.
5-13. One exemplary expression vector is pEGEN 1.1 shown in FIG. 5.
As shown, pEGEN 1.1 comprises an SV40 promoter sequence (1), an
intronic sequence (2), a multiple cloning sequence (MCS), a human
growth hormone poly-A tail sequence (4), a kanamycin resistance
gene (7) and a pUC origin of replication (8). DNA fragments
encoding for Sec-RNA molecules or fusion proteins are prepared by
PCR with primers including restriction sites for subcloning into
the multiple cloning sequence. PCR products and the pEGEN1.1
plasmid are digested with the appropriate restriction enzymes and
purified prior to ligation. Sec-RNA molecules or mRNAs encoding
fusion proteins are transcribed from the SV40 promoter sequence
with an artificial intron and polyA tail sequence.
[0152] Another exemplary expression vector is pEGEN 2.1 shown in
FIG. 6. As shown, pEGEN 2.1 comprises a chicken .beta.-actin
promoter sequence (1), an intronic sequence (2), a multiple cloning
sequence (MCS), a human growth hormone poly-A tail sequence (4), a
kanamycin resistance gene (7) and a pUC origin of replication (8).
DNA fragments encoding for Sec-RNA molecules or fusion proteins are
prepared by PCR with primers including restriction sites for
subcloning into the multiple cloning sequence. PCR products and the
pEGEN2.1 plasmid are digested with the appropriate restriction
enzymes and purified prior to ligation. Sec-RNA molecules or mRNAs
encoding fusion proteins are transcribed from the chicken-actin
promoter sequence with an artificial intron and polyA tail
sequence.
[0153] Another exemplary expression vector is pEGEN 3.1 shown in
FIG. 7. As shown, pEGEN 3.1 comprises a CMV promoter sequence (1),
an intronic sequence (2), a multiple cloning sequence (MCS), a
human growth hormone poly-A tail sequence (4), a kanamycin
resistance gene (7) and a pUC origin of replication (8). DNA
fragments encoding for Sec-RNA molecules or fusion proteins are
prepared by PCR with primers including restriction sites for
subcloning into the multiple cloning sequence. PCR products and the
pEGEN3.1 plasmid are digested with the appropriate restriction
enzymes and purified prior to ligation. Sec-RNA molecules or mRNAs
encoding fusion proteins are transcribed from the CMV promoter
sequence with an artificial intron and polyA tail sequence.
[0154] Another exemplary expression vector is pEGEN 4.1 shown in
FIG. 8. As shown, pEGEN 4.1 comprises a human U6 promoter sequence
(1), a multiple cloning sequence (MCS), a polyT terminator sequence
(4), a kanamycin resistance gene (7) and a pUC origin of
replication (8). DNA fragments encoding for Sec-RNA molecules are
prepared by PCR with primers including restriction sites for
subcloning into the multiple cloning sequence. PCR products and the
pEGEN4.1 plasmid are digested with the appropriate restriction
enzymes and purified prior to ligation. Sec-RNA molecules are
transcribed from the U6 promoter sequence and terminate with the
polyT terminator sequence.
[0155] Another exemplary expression vector is pBioR Pol II (shown
in FIG. 9) which encodes an exemplary RNA-protein complex of the
invention. The vector comprises an SV40 promoter (1) upstream of an
Sec-RNA sequence (3) and a downstream hGH polyA sequence (4). The
vector also comprises a .beta.-actin promoter (5) upstream of a
fusion protein sequence (6) and a downstream hGH polyA sequence
(4). The vector also comprises a kanamycin resistance gene (7) and
a pUC origin of replication (8).
[0156] Another exemplary expression vector is pBioR Pol III shown
in FIG. 10 which encodes an exemplary RNA-protein complex of the
invention. The vector comprises an hU6 promoter upstream (1) of an
Sec-RNA sequence (3) and a downstream Pol-III poly-T terminator
sequence (4). The vector also comprises a .beta.-actin promoter (5)
upstream of a fusion protein sequence (6) and a downstream hGH
polyA sequence (4). The vector also comprises a kanamycin
resistance gene (7) and a pUC origin of replication (8).
[0157] Another exemplary expression vector is pBioR Pol II combo
shown in FIG. 11 which encodes an exemplary RNA-protein complex of
the invention. The vector comprises a .beta.-actin promoter (1), an
intronic sequence (2), a fusion protein (6), a Sec-RNA (3) with
flanking introns (2) internal to the fusion protein, a human growth
hormone poly-A tail sequence (4), a kanamycin resistance gene (7)
and a pUC origin of replication (8). In this expression vector, the
Sec-RNA is encoded within an artificial intron placed within the
mRNA sequence encoding the fusion protein. DNA fragments encoding
for Sec-RNA molecules or fusion proteins are prepared by PCR. DNA
fragments encoding for Sec-RNA molecules are prepared with primers
including splice donor and acceptor sites and restriction sites for
subcloning into a unique restriction site within the fusion protein
sequence. DNA fragments encoding for the fusion protein are
prepared with primers including restriction sites for subcloning
into the plasmids described above. After transcription, the Sec-RNA
is released from the mRNA encoding the fusion protein by the
splicing machinery endogenous to the bioreactor cell.
[0158] Another exemplary expression vector is pBioR Pol II stable
shown in FIG. 12 which encodes an exemplary RNA-protein complex of
the invention. The vector comprises a CTS regulator (9), a PGK
promoter (1), a puromycin resistance gene (10), a chicken
.beta.-actin promoter (5), a fusion protein (6), a Sec-RNA (3) with
flanking introns (2) internal to the fusion protein, a human growth
hormone poly-A tail sequence (4), a kanamycin resistance gene (7)
and a pUC origin of replication (8). Sec-RNA sequences can be
selected from Tables I and II; fusion protein sequences can be
selected from Tables III, IV and V.
[0159] Another exemplary expression vector is pBioR Pol II dicer
shown in FIG. 13 which encodes an exemplary RNA-protein complex of
the invention. The vector comprises a SV40 promoter (1), an
intronic sequence (2), a biologically active RNA sequence and a
recognition RNA sequence (3), a hGH poly-A tail sequence (4), a
chicken-actin promoter (5), a fusion protein (6), a Sec-RNA (3)
with flanking introns (2) internal to the fusion protein, a human
growth hormone poly-A tail sequence (4), a kanamycin resistance
gene (7) and a pUC origin of replication (8). Sec-RNA sequences can
be selected from Tables I and II; fusion protein sequences can be
selected from Tables III, IV and V.
[0160] In other embodiments, the expression vector comprises a
first polynucleotide sequence that encodes a nucleic acid molecule
comprising one or more biologically active RNA sequences, a
recognition RNA sequence, and optionally a terminal minihelix
sequence and a second polynucleotide sequence that encodes a
polypeptide comprising an RNA binding domain, and one or more
transport peptide sequences. In another embodiment, the expression
vector further comprises a third polynucleotide that encodes a
nucleic acid molecule comprising one or more biologically active
RNA sequences, optionally a recognition RNA sequence, and
optionally a terminal minihelix sequence. In one embodiment, the
biologically active RNAs of the first polynucleotide and the third
polynucleotide are targeted to one or more target genes of
interest. In another embodiment, the biologically active RNA of the
first polynucleotide is selected from a short interfering RNA
(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short
hairpin RNA (shRNA) targeted to one or more target genes of
interest and the biologically active RNA of the third
polynucleotide is targeted to Dicer and/or Drosha.
[0161] In a further embodiment, the expression vector additionally
comprises a first promoter sequence, a termination sequence, and
optionally one or more primers sequences, a second promoter
sequence, a polyA addition sequence, and optionally one or more
primers sequences, wherein the first polynucleotide encoding the
one or more biologically active RNA sequences, the recognition RNA
sequence, and the optional terminal minihelix sequence is operably
linked to the first promoter sequence and the termination sequence
and wherein the second polynucleotide encoding the RNA binding
domain sequence and the transport peptide sequence is operably
linked to the second promoter sequence and the polyA addition
sequence. In addition, the vector can additionally comprises one or
more promoter sequences, one or more termination sequences, and
optionally one or more primers sequences, wherein the third
polynucleotide sequence(s) encoding the nucleic acid comprising one
or more biologically active RNA sequences, optionally a recognition
RNA sequence, and optionally a terminal minihelix sequence is
operably linked to the one or more promoter sequences and the one
or more termination sequences.
[0162] In another embodiment, the expression vector further
comprises one or more polynucleotide sequences encoding one or more
viral polymerases and one or more viral accessory proteins
necessary for viral replication. In a further embodiment, the
vector additionally comprises one or more promoter sequences, one
or more polyA addition sequences, and optionally one or more
primers sequences, wherein the polynucleotide sequence(s) encoding
the viral polymerase(s) and the viral accessory protein(s) is
operably linked to the one or more promoter sequences and the one
or more polyA addition sequences.
[0163] In one embodiment, the invention provides an expression
vector comprising a polynucleotide that encodes a nucleic acid
molecule comprising one or more biologically active RNA sequences,
a recognition RNA sequence, and an optional terminal minihelix
sequence. In one embodiment, the expression vector comprises a
polynucleotide that encodes a nucleic acid molecule comprising one
or more biologically active RNA sequences and one or more
polynucleotide sequences encoding one or more viral polymerases and
one or more viral accessory proteins necessary for viral
replication.
[0164] The invention also provides an expression vector comprising
a polynucleotide that encodes a polypeptide comprising an RNA
binding domain and one or more transport peptides.
[0165] Thus, the invention provides a first expression vector
comprising a polynucleotide that encodes a nucleic acid molecule
comprising one or more biologically active RNA sequences, a
recognition RNA sequence and optionally a terminal minihelix
sequence and a second expression vector comprising a polynucleotide
that encodes a polypeptide comprising an RNA binding domain and one
or more transport peptides, for example, a peptide selected from a
cell penetrating peptide, a non-classical secretory domain, a
receptor binding domain, a fusogenic peptide, and an endosomal
release domain.
[0166] In any of the expression vectors of the invention, one or
more of the sequences comprising the recognition RNA sequence, the
individual biologically active RNA sequences, the optional terminal
minihelix sequence, the RNA binding domain, and the transport
peptide(s), as well as any other sequences, including viral
sequences, promoters, primers, termination sequences, and polyA
sequences are joined directly without the addition of one or more
intervening or additional sequences. Alternatively, one or more of
the sequences comprising the recognition RNA sequence, the
individual biologically active RNA sequences, the optional terminal
minihelix sequence, the RNA binding domain, and the transport
peptide(s), as well as any other sequences, including viral
sequences, promoters, primers, termination sequences, and polyA
sequences are joined with the addition of one or more intervening
or additional sequences. In any of the above-described embodiments,
the individual biologically active RNA sequences themselves are
joined directly without any intervening or additional sequences or
are joined with the addition of one or more intervening or
additional sequences. In any of the above-described embodiments,
the recognition RNA sequence and any of the biologically active
RNAs are joined directly without the addition of one or more
linker, spacer, or other sequences or are joined with the addition
of one or more linker, spacer, and/or other sequences. In any of
the above-described embodiments, the RNA binding domain and any of
the individual transport peptides are joined directly without the
addition of one or more linker, spacer, or other sequences or are
joined with the addition of one or more linker, spacer, and/or
other sequences.
[0167] In certain embodiments of the described expression vectors,
the biologically active RNA sequence is selected from a ribozyme,
antisense nucleic acid, allozyme, aptamer, short interfering RNA
(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short
hairpin RNA (shRNA), and a transcript encoding one or more
biologically active peptides. In one specific embodiment, the
biologically active RNA sequence is a short hairpin RNA (shRNA). In
another specific embodiment, the biologically active RNA sequence
is an aptamer. In certain embodiments, the recognition RNA sequence
is selected from a U1 loop, Group II intron, NRE stem loop, S1A
stem loop, Bacteriophage BoxBR, HIV Rev response element, AMVCP
recognition sequence, and ARE sequence. In one embodiment, the
terminal minihelix sequence is from the adenovirus VA1 RNA
molecule. In certain embodiments, the RNA binding domain is
selected from a U1A, CRS1, CRM1, Nucleolin RBD12, hRBMY,
Bacteriophage Protein N, HIV Rev, alfalfa mosaic virus coat protein
(AMVCP), and tristetrapolin amino acid sequence. In certain
embodiments, the one or more transport peptides is selected from a
cell penetrating peptide, a non-classical secretory domain, a
receptor binding domain, a fusogenic peptide, and an endosomal
release domain, as well as any combinations thereof. In one
specific embodiment, the transport peptide is a cell penetrating
peptide. In certain specific embodiments, the cell penetrating
peptide is selected from a penetratin, transportan, MAP, HIV TAT,
Antp, Rev, FHV coat protein, TP10, and pVEC sequence. In another
specific embodiment, the transport peptide is a non-classical
secretory domain. In certain specific embodiments, the
non-classical secretory domain is selected from a Galcetin-1
peptide, Galectin-3 peptide, IL-1.alpha., IL-1.beta., HASPB, HMGB1,
FGF-1, FGF-2, IL-2 signal, secretory transglutaminase, annexin-1,
HIV TAT, Herpes VP22, thioredoxin, Rhodanese, and plasminogen
activator signal sequence. In one specific embodiment, the
transport peptides are a cell penetrating peptide, and one or more
transport peptides selected from a non-classical secretory domain,
a receptor binding domain, a fusogenic peptide, and an endosomal
release domain. In one specific embodiment, the transport peptides
are a cell penetrating peptide, and a non-classical secretory
domain. In certain embodiments, the viral non-structural and
structural genes (viral polymerases, accessory proteins, coat
proteins, and fusogenic proteins) are selected from DNA viruses and
RNA viruses, including, but not limited to, Adenovirus,
Adeno-Associated Virus, Herpes Simplex Virus Lentivirus,
Retrovirus, Sindbis virus, and Foamy virus.
[0168] In addition the present invention provides expression
vectors constructed from a replication competent or replication
incompetent viral particles which carry and distribute one or more
biologically active RNA molecules from a transformed packaging
cell. In one embodiment, the invention provides a viral vector
comprising a partial viral genome and a second viral vector
comprising a partial viral genome and a polynucleotide that encodes
any of the nucleic acid molecules described herein. In one
embodiment, the invention provides a viral vector comprising a
polynucleotide that encodes a nucleic acid molecule comprising one
or more biologically active RNA sequences, a recognition RNA
sequence, and optionally a terminal minihelix sequence and a
polynucleotide that encodes a polypeptide comprising one or more
fusion proteins, ie. RNA binding domain and one or more transport
peptides. The biologically active RNA sequence can be any of the
biologically active RNA sequences described herein and otherwise
known in the art. In one embodiment, the viral vector comprises a
polynucleotide encoding a nucleic acid molecule wherein the
biologically active RNA sequence is selected from a ribozyme,
antisense nucleic acid, allozyme, aptamer, short interfering RNA
(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short
hairpin RNA (shRNA), and a transcript encoding one or more
biologically active peptides. In one specific embodiment, the viral
vector comprises a polynucleotide encoding a nucleic acid molecule
wherein the biologically active RNA sequence is a short hairpin RNA
(shRNA). In one specific embodiment, the viral vector comprises a
polynucleotide encoding a nucleic acid molecule wherein the
biologically active RNA sequence is an aptamer. The recognition RNA
sequence can be any of the recognition RNA sequences described
herein and otherwise known in the art. In one embodiment, viral
vector vector comprises a polynucleotide encoding a nucleic acid
molecule wherein the recognition RNA sequence is selected from a U1
loop, Group II intron, NRE stem loop, S1A stem loop, Bacteriophage
BoxBR, HIV Rev response element, AMVCP recognition sequence, and
ARE sequence. The terminal minihelix sequence can be any of the
terminal minimhelix sequences described herein and otherwise known
in the art. The invention also provides an expression vector
comprising a polynucleotide that encodes a polypeptide comprising
an RNA binding domain and one or more transport peptides. In
certain embodiments, the RNA binding domain is selected from a U1A,
CRS1, CRM1, Nucleolin RBD12, hRBMY, Bacteriophage Protein N, HIV
Rev, alfalfa mosaic virus coat protein (AMVCP), and tristetrapolin
amino acid sequence. In certain embodiments, the one or more
transport peptides is selected from a cell penetrating peptide, a
non-classical secretory domain, a receptor binding domain, a
fusogenic peptide, and an endosomal release domain, as well as any
combinations thereof. In one embodiment, the invention provides an
expression vector comprising a polynucleotide that encodes a
polypeptide comprising an RNA binding domain and a cell penetrating
peptide. In certain specific embodiments, the cell penetrating
peptide is selected from a penetratin, transportan, MAP, HIV TAT,
Antp, Rev, FHV coat protein, TP10, and pVEC sequence. In another
embodiment, the invention provides an expression vector comprising
a polynucleotide that encodes a polypeptide comprising an RNA
binding domain and a non-classical secretory domain. In certain
specific embodiments, the non-classical secretory domain is
selected from a Galcetin-1 peptide, Galectin-3 peptide,
IL-1.alpha., IL-1.beta., HASPB, HMGB1, FGF-1, FGF-2, IL-2 signal,
secretory transglutaminase, annexin-1, HIV TAT, Herpes VP22,
thioredoxin, Rhodanese, and plasminogen activator signal sequence.
In one embodiment, the invention provides an expression vector
comprising a polynucleotide that encodes a polypeptide comprising
an RNA binding domain, a cell penetrating peptide, and one or more
transport peptides selected from a non-classical secretory domain,
a receptor binding domain, a fusogenic peptide, and an endosomal
release domain. In one embodiment, the invention provides an
expression vector comprising a polynucleotide that encodes a
polypeptide comprising an RNA binding domain, a cell penetrating
peptide, and a non-classical secretory domain.
[0169] In another embodiment, the viral vector additionally
comprises a polynucleotide that encodes a partial viral genome and
a nucleic acid molecule comprising one or more biologically active
RNA sequences targeted to Dicer and/or Drosha. None of these
polynucleotides encode an RNA binding domain. In one embodiment,
the polynucleotide encodes a nucleic acid molecule comprising a
single biologically active RNA sequence. In another embodiment, the
polynucleotide encodes a nucleic acid molecule comprising two or
more biologically active RNA sequences. In certain embodiments, the
biologically active RNA sequence is selected from a ribozyme,
antisense nucleic acid, allozyme, aptamer, short interfering RNA
(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short
hairpin RNA (shRNA), and a transcript encoding one or more
biologically active peptides.
[0170] Bioreactor Cells
[0171] BioReactor cells are generated by transfecting an expression
vector of the invention, for example a pBioR plasmid or plasmids,
into a recipient cell line in vitro. Any cell type can serve as a
recipient cell for the expression vector(s), including any of the
pBioR plasmids. The source of the potential BioReactor cell can
vary depending on the identity of domains used in the fusion
protein and the identity of the cells being targeted for gene
knockdown. BioReactors are capable of producing the fusion protein,
producing the Sec-RNA, binding of the Sec-RNA by the fusion protein
and secretion of the RNA-protein complex. Production of the fusion
protein can be verified through RT-PCR based assays that detect the
plasmid derived mRNA transcript encoding the protein and antibody
based assays that detect the protein itself. Successful production
of the Sec-RNA includes both transcription of the RNA (biologically
active RNA and recognition RNA sequence) and export of that
transcript from the nucleus. RT-PCR assays can be used to show
production of the plasmid derived Sec-RNA molecule and cellular
fractionation can be used to demonstrate accumulation of the RNA in
the cytoplasm. Binding of the Sec-RNA molecule by the fusion
protein can be demonstrated by immunoprecipitation of the
RNA-protein complex using an antibody to one of the domains of the
fusion protein or, alternatively, via the addition of an epitope
tag (FLAG, HA, etc.) to the fusion protein sequence. Secretion of
the RNA-protein complex can be verified by detection of the Sec-RNA
in the extracellular matrix, or media in the case of cells in
culture. Intact RNA-protein complexes can be isolated from the
media via immunoprecipitation, as described above, or total RNA may
be prepped using Tri-Reagent (Sigma-Aldrich, product #T9424). The
Sec-RNA is detected by northern blotting or by RT-PCR as described
above.
[0172] The BioReactor cells can be produced by transient
transfection of a suitable cell with an expression vector of the
invention or by the development of stably transfected cells, where
the plasmid is integrated into the genome of the BioReactor cell.
Cells can be transiently transfected with an expression vector of
the invention via liposomal or polymeric delivery agents or via
electroporation using methods described herein and otherwise known
in the art. The efficiency of these types of transfection precludes
the need for purification of BioReactor cells (i.e., transfected
cells) from non-transfected cells, which behaves as inert starting
material in subsequent delivery steps. In contrast, the development
of cell lines stably transfected with an expression vector of the
invention and expressing the fusion protein and Sec-RNA from the
genome of the recipient cell requires isolation of individual
colonies of transfected cells, each representing a single
integration event and giving rise to a homogeneous population of
BioReactor cells. These cells produce secretion complexes
continuously and are useful in both in vitro and in vivo
applications.
[0173] Bioreactor cells can be used as transfection agents to
facilitate the delivery of the Sec-RNA molecule to cells.
Bioreactor cells can also be applied to target cells in vitro, ex
vivo, or in vivo for the purpose of knocking down the gene product
targeted by the Sec-RNA molecule. The particular expression vector
and recipient cells used in the transfection are determined by the
gene target of interest and the target cell identity. Likewise, the
optimal ratio of BioReactor cells to target cells is determined
empirically for each system of cells and gene targets. RNA and/or
protein samples are collected from the target cells about 24-72
hours after addition of the BioReactor cells in order to assay
knockdown of the mRNA transcript or protein, respectively. The mRNA
levels of the target gene can be measured via RT-PCR, Northern blot
and other methods known in the art. The protein levels of the
target gene can be measured using known methods such as Western
blot and immunoprecipitation.
[0174] Bioreactor cells can be generated by administering one or
more of the expression vectors of the invention. In one embodiment,
the invention provides a bioreactor cell comprising any of the
expression vectors and compositions thereof provided herein. In one
embodiment, the invention provides a cell comprising an expression
vector comprising a polynucleotide sequence encoding a nucleic acid
comprising a biologically active RNA sequence, a recognition RNA
sequence, and optionally a terminal minihelix sequence and a
polynucleotide sequence encoding a polypeptide comprising an RNA
binding domain sequence and a transport peptide.
[0175] In one embodiment, the invention provides a cell comprising
an expression vector comprising a polynucleotide sequence encoding
a nucleic acid comprising a biologically active RNA sequence, a
recognition RNA sequence, and optionally a terminal minihelix
sequence, a polynucleotide sequence encoding a polypeptide
comprising an RNA binding domain sequence and a transport peptide,
and one or more polynucleotide sequences encoding one or more viral
polymerases and one or more viral accessory proteins necessary for
viral replication and an expression vector comprising one or more
polynucleotide sequences encoding one or more viral coat proteins
and one or more viral fusogenic proteins.
[0176] In one embodiment, the invention provides a cell comprising
an expression vector comprising a polynucleotide sequence encoding
a nucleic acid comprising a biologically active RNA sequence, a
recognition RNA sequence, and optionally a terminal minihelix
sequence, a polynucleotide sequence encoding a polypeptide
comprising an RNA binding domain sequence and a transport peptide,
and an additional polynucleotide sequence encoding a nucleic acid
comprising one or more biologically active RNA sequences that
target one or more further gene target(s). In one embodiment, the
additional polynucleotide sequence encodes a nucleic acid
comprising one or more biologically active RNA sequences that
target a further gene target and an RNA recognition sequence. In
another embodiment, where one of the biologically active RNA
sequences in the vector is a short interfering RNA (siRNA),
double-stranded RNA (dsRNA), micro-RNA (miRNA), or short hairpin
RNA (shRNA), the additional polynucleotide sequence encodes a
nucleic acid comprising one or more biologically active RNA
sequences targeted to Dicer and/or Drosha.
[0177] In one embodiment, the invention provides a cell comprising
an expression vector comprising a polynucleotide sequence encoding
a nucleic acid comprising a biologically active RNA sequence, a
recognition RNA sequence, and optionally a terminal minihelix
sequence, a polynucleotide sequence encoding a polypeptide
comprising an RNA binding domain sequence and a transport peptide,
one or more polynucleotide sequences encoding one or more viral
polymerases and one or more viral accessory proteins necessary for
viral replication, and an additional polynucleotide sequence
encoding a nucleic acid comprising one or more biologically active
RNA sequences that target one or more further gene target(s) (for
example, Dicer and/or Drosha gene targets) and an expression vector
comprising one or more polynucleotide sequences encoding one or
more viral coat proteins and one or more viral fusogenic
proteins.
[0178] In one embodiment, the invention provides a cell comprising
an expression vector comprising a polynucleotide sequence encoding
a nucleic acid comprising a biologically active RNA sequence and
one or more polynucleotide sequences encoding one or more viral
polymerases and one or more viral accessory proteins necessary for
viral replication, and an expression vector comprising one or more
polynucleotide sequences encoding one or more viral coat proteins
and one or more viral fusogenic proteins.
[0179] In one embodiment, the invention provides a cell comprising
an expression vector comprising a polynucleotide sequence encoding
a nucleic acid comprising a biologically active RNA sequence, a
recognition RNA sequence, and optionally a terminal minihelix
sequence and an expression vector comprising a polynucleotide
sequence encoding a polypeptide comprising an RNA binding domain
sequence and one or more transport peptides. In one embodiment, the
cell further comprises a third expression vector comprising a
polynucleotide sequence encoding a nucleic acid comprising one or
more biologically active RNA sequences that target one or more gene
target(s) that differ from the gene target(s) of the biologically
active RNA in the first expression vector. In one embodiment, the
third expression vector comprises a polynucleotide sequence
encoding a nucleic acid comprising one or more biologically active
RNA sequences that target one or more gene targets and an RNA
recognition sequence. In another embodiment, where one of the
biologically active RNA sequences in the first expression vector is
a short interfering RNA (siRNA), double-stranded RNA (dsRNA),
micro-RNA (miRNA), or short hairpin RNA (shRNA), the third
expression vector comprises a polynucleotide sequence encoding a
nucleic acid comprising one or more biologically active RNA
sequences targeted to Dicer and/or Drosha.
[0180] The bioreactor cells described herein can be used, among
other things, to deliver biologically active RNA to target cells.
In one embodiment, the method of delivering a biologically active
RNA to target cells comprises the steps of: (a) preparing an
expression vector that encodes an RNA-protein complex comprising
one or more biologically active RNAs, a recognition RNA sequence,
optionally a terminal minihelix sequence, an RNA binding domain,
and one or more transport peptide sequences selected from a cell
penetrating domain, non-classical secretory domain, endosomal
release domain, fusogenic peptide and a receptor binding domain;
(b) administering the expression vector of step (a) to cells in
culture to produce bioreactor cells expressing the RNA-protein
complex; (c) collecting the cultured cells of step (b); (d) testing
the cells of (c) to determine the bioreactor cells expressing the
RNA-protein complex; and (e) isolating the bioreactor cells from
the other cells in culture; and (f) mixing one or more target cells
with the isolated bioreactor cells in step (e) to deliver a
biologically active RNA to the target cells. In one embodiment, the
target cells of (f) are target cells in cell culture. In one
embodiment, the target cells of (f) are target cells extracted from
an organism, including a mammalian animal. In one embodiment, the
mammalian animal is a human. The expression vector can be any
expression vector described herein. The RNA-protein complex can be
any RNA-protein complex described herein. In one embodiment, the
biologically active RNA of the RNA-protein complex is an shRNA. In
another embodiment, the biologically active RNA of the RNA-protein
complex is an aptamer. In one embodiment, the cells of step (b) are
stably transfected with the expression vector. In certain
embodiments of the method, the expression vector of step (a)
further comprises a polynucleotide sequence encoding a nucleic acid
comprising one or more biologically active RNA sequences that
target one or more further gene target(s). In one embodiment, the
additional polynucleotide sequence encodes a nucleic acid
comprising one or more biologically active RNA sequences that
target a further gene target and an RNA recognition sequence. In
another embodiment, where one of the biologically active RNA
sequences in the vector is a short interfering RNA (siRNA),
double-stranded RNA (dsRNA), micro-RNA (miRNA), or short hairpin
RNA (shRNA), the additional polynucleotide sequence encodes a
nucleic acid comprising one or more biologically active RNA
sequences targeted to Dicer and/or Drosha.
[0181] In another embodiment, the method of delivering a
biologically active RNA to target cells comprises the steps of: (a)
preparing an expression vector comprising a polynucleotide sequence
encoding nucleic acid comprising one or more biologically active
RNAs, a recognition RNA sequence, and optionally a terminal
minihelix sequence, a polynucleotide sequence encoding a
polypeptide comprising an RNA binding domain, and one or more
transport peptide sequences, and one or more polynucleotide
sequences encoding one or more viral polymerases and one or more
viral accessory proteins necessary for viral replication; (b)
preparing an expression vector comprising one or more
polynucleotide sequences encoding one or more viral coat proteins
and one or more viral fusogenic proteins; (c) administering the
expression vector of step (a) and the expression vector of (b) to
cells in culture to produce bioreactor cells expressing the
RNA-protein complex; (d) collecting the cultured cells of step (c);
(e) testing the cells of (d) to determine the bioreactor cells
expressing the RNA-protein complex; and (f) isolating the
bioreactor cells from the other cells in culture; and (g) mixing
one or more target cells with the isolated bioreactor cells in step
(f) to deliver a biologically active RNA to the target cells. In
one embodiment, the target cells of (g) are target cells in cell
culture. In one embodiment, the target cells of (g) are target
cells extracted from an organism, including a mammalian animal. In
one embodiment, the mammalian animal is a human. In one embodiment,
the cells of step (c) are stably transfected with the expression
vector.
[0182] In certain embodiments of the method, the expression vector
of step (a) further comprises a polynucleotide sequence encoding a
nucleic acid comprising one or more biologically active RNA
sequences that target one or more further gene target(s). In one
embodiment, the additional polynucleotide sequence encodes a
nucleic acid comprising one or more biologically active RNA
sequences that target a further gene target and an RNA recognition
sequence. In another embodiment, where one of the biologically
active RNA sequences in the vector is a short interfering RNA
(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), or short
hairpin RNA (shRNA), the additional polynucleotide sequence encodes
a nucleic acid comprising one or more biologically active RNA
sequences targeted to Dicer and/or Drosha.
[0183] In another embodiment, the method of delivering a
biologically active RNA to target cells comprises the steps of: (a)
preparing an expression vector comprising a polynucleotide sequence
encoding nucleic acid comprising one or more biologically active
RNAs and one or more polynucleotide sequences encoding one or more
viral polymerases and one or more viral accessory proteins
necessary for viral replication; (b) preparing an expression vector
comprising one or more polynucleotide sequences encoding one or
more viral coat proteins and one or more viral fusogenic proteins;
(c) administering the expression vector of step (a) and the
expression vector of (b) to cells in culture to produce bioreactor
cells expressing the RNA-protein complex; (d) collecting the
cultured cells of step (c); (e) testing the cells of (d) to
determine the bioreactor cells expressing the RNA-protein complex;
and (f) isolating the bioreactor cells from the other cells in
culture; and (g) mixing one or more target cells with the isolated
bioreactor cells in step (f) to deliver a biologically active RNA
to the target cells. In one embodiment, the target cells of (g) are
target cells in cell culture. In one embodiment, the target cells
of (g) are target cells extracted from an organism, including a
mammalian animal. In one embodiment, the mammalian animal is a
human. In one embodiment, the cells of step (c) are stably
transfected with the expression vector.
[0184] In another embodiment, the method of delivering a
biologically active RNA to target cells comprises the steps of: (a)
preparing an expression vector comprising a polynucleotide sequence
encoding nucleic acid comprising one or more biologically active
RNAs, a recognition RNA sequence, and optionally a terminal
minihelix sequence; (b) preparing an expression vector comprising a
polynucleotide sequence encoding a polypeptide comprising an RNAs
binding domain and one or more transport peptides; (c)
administering the expression vector of step (a) and the expression
vector of (b) to cells in culture to produce bioreactor cells
expressing the RNA-protein complex; (d) collecting the cultured
cells of step (c); (e) testing the cells of (d) to determine the
bioreactor cells expressing the RNA-protein complex; and (f)
isolating the bioreactor cells from the other cells in culture; and
(g) mixing one or more target cells with the isolated bioreactor
cells in step (f) to deliver a biologically active RNA to the
target cells. In one embodiment, the target cells of (g) are target
cells in cell culture. In one embodiment, the target cells of (g)
are target cells extracted from an organism, including a mammalian
animal. In one embodiment, the mammalian animal is a human. In one
embodiment, the cells of step (c) are stably transfected with the
expression vector.
[0185] In another embodiment, the methods comprises preparing and
administering a third expression vector comprising a polynucleotide
sequence encoding a nucleic acid comprising one or more
biologically active RNA sequences that target one or more further
gene target(s). In one embodiment, the additional polynucleotide
sequence encodes a nucleic acid comprising one or more biologically
active RNA sequences that target a further gene target and an RNA
recognition sequence. In another embodiment, where one of the
biologically active RNA sequences in the first vector is a short
interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA
(miRNA), or short hairpin RNA (shRNA), the additional
polynucleotide sequence encodes a nucleic acid comprising one or
more biologically active RNA sequences targeted to Dicer and/or
Drosha.
[0186] The invention also provides methods of using the bioreactor
cells ex vivo for the delivery of a biologically active RNA to
target cells. In one embodiment, the method of delivering a
biologically active RNA to target cells ex vivo comprises the steps
of: (a) preparing an expression vector that encodes an RNA-protein
complex comprising one or more biologically active RNAs, a
recognition RNA sequence, optionally a terminal minihelix sequence,
an RNA binding domain, and one or more target peptide sequences;
(b) administering the expression vector of step (a) to cells in
culture to produce bioreactor cells expressing the RNA-protein
complex; (c) collecting the cultured cells of step (b); (d)
obtaining target cells from a subject; (e) mixing one or more
target cells obtained in step (d) with the cultured cell(s)
collected in step (c) to deliver a biologically active RNA to the
target cells. In one embodiment, the method further comprises the
step of (f) administering the cells in step (e) to a subject. In
one embodiment, the method further comprises the step of separating
the bioreactor cells from the target cells before administering the
target cells to the subject. In one embodiment, the subject of step
(f) is the same subject as the subject in step (d) from which the
target cells were obtained. In another embodiment, the subject of
step (f) is a different subject from the subject in step (d) from
which the target cells were obtained. In certain embodiments of the
method, the expression vector of step (a) further comprises a
polynucleotide sequence encoding a nucleic acid comprising one or
more biologically active RNA sequences that target one or more
further gene target(s). In one embodiment, the additional
polynucleotide sequence encodes a nucleic acid comprising one or
more biologically active RNA sequences that target a further gene
target and an RNA recognition sequence. In another embodiment,
where one of the biologically active RNA sequences in the vector is
a short interfering RNA (siRNA), double-stranded RNA (dsRNA),
micro-RNA (miRNA), or short hairpin RNA (shRNA), the additional
polynucleotide sequence encodes a nucleic acid comprising one or
more biologically active RNA sequences targeted to Dicer and/or
Drosha.
[0187] In another embodiment, the method of delivering a
biologically active RNA to target cells comprises the steps of: (a)
preparing an expression vector comprising a polynucleotide sequence
encoding nucleic acid comprising one or more biologically active
RNAs, a recognition RNA sequence, and optionally a terminal
minihelix sequence, a polynucleotide sequence encoding a
polypeptide comprising an RNA binding domain, and one or more
transport peptide sequences, and one or more polynucleotide
sequences encoding one or more viral polymerases and one or more
viral accessory proteins necessary for viral replication; (b)
preparing an expression vector comprising one or more
polynucleotide sequences encoding one or more viral coat proteins
and one or more viral fusogenic proteins; (c) administering the
expression vector of step (a) and the expression vector of (b) to
cells in culture to produce bioreactor cells expressing the
RNA-protein complex; (d) collecting the cultured cells of step (c);
(e) obtaining target cells from a subject; (f) mixing one or more
target cells obtained in step (e) with the cultured cell(s)
collected in step (d) to deliver a biologically active RNA to the
target cells. In one embodiment, the method further comprises the
step of: (g) administering the cells in step (d) to a subject. In
one embodiment, the method further comprises the step of separating
the bioreactor cells from the target cells before administering the
target cells to the subject. In one embodiment, the subject of step
(g) is the same subject as the subject in step (e) from which the
target cells were obtained. In another embodiment, the subject of
step (g) is a different subject from the subject in step (e) from
which the target cells were obtained.
[0188] In certain embodiments of the method, the expression vector
of step (a) further comprises a polynucleotide sequence encoding a
nucleic acid comprising one or more biologically active RNA
sequences that target one or more further gene target(s). In one
embodiment, the additional polynucleotide sequence encodes a
nucleic acid comprising one or more biologically active RNA
sequences that target a further gene target and an RNA recognition
sequence. In another embodiment, where one of the biologically
active RNA sequences in the vector is a short interfering RNA
(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), or short
hairpin RNA (shRNA), the additional polynucleotide sequence encodes
a nucleic acid comprising one or more biologically active RNA
sequences targeted to Dicer and/or Drosha.
[0189] In another embodiment, the method of delivering a
biologically active RNA to target cells comprises the steps of: (a)
preparing an expression vector comprising a polynucleotide sequence
encoding nucleic acid comprising one or more biologically active
RNAs and one or more polynucleotide sequences encoding one or more
viral polymerases and one or more viral accessory proteins
necessary for viral replication; (b) preparing an expression vector
comprising one or more polynucleotide sequences encoding one or
more viral coat proteins and one or more viral fusogenic proteins;
(c) administering the expression vector of step (a) and the
expression vector of (b) to cells in culture to produce bioreactor
cells expressing the RNA-protein complex; (d) collecting the
cultured cells of step (c); (e) obtaining target cells from a
subject; (f) mixing one or more target cells obtained in step (e)
with the cultured cell(s) collected in step (d) to deliver a
biologically active RNA to the target cells. In one embodiment, the
method further comprises the step of: (g) administering the cells
in step (d) to a subject. In one embodiment, the method further
comprises the step of separating the bioreactor cells from the
target cells before administering the target cells to the subject.
In one embodiment, the subject of step (g) is the same subject as
the subject in step (e) from which the target cells were obtained.
In another embodiment, the subject of step (g) is a different
subject from the subject in step (e) from which the target cells
were obtained.
[0190] In another embodiment, the method of delivering a
biologically active RNA to target cells comprises the steps of: (a)
preparing an expression vector comprising a polynucleotide sequence
encoding nucleic acid comprising one or more biologically active
RNAs, a recognition RNA sequence, and optionally a terminal
minihelix sequence; (b) preparing an expression vector comprising a
polynucleotide sequence encoding a polypeptide comprising an RNAs
binding domain and one or more transport peptides; (c)
administering the expression vector of step (a) and the expression
vector of (b) to cells in culture to produce bioreactor cells
expressing the RNA-protein complex; (d) collecting the cultured
cells of step (c); (e) obtaining target cells from a subject; (f)
mixing one or more target cells obtained in step (e) with the
cultured cell(s) collected in step (d) to deliver a biologically
active RNA to the target cells. In one embodiment, the method
further comprises the step of: (g) administering the cells in step
(d) to a subject. In one embodiment, the method further comprises
the step of separating the bioreactor cells from the target cells
before administering the target cells to the subject. In one
embodiment, the subject of step (g) is the same subject as the
subject in step (e) from which the target cells were obtained. In
another embodiment, the subject of step (g) is a different subject
from the subject in step (e) from which the target cells were
obtained.
[0191] In any of the above described methods, the method can
further comprise the steps of: testing the cells of (c) or (d) to
determine the bioreactor cells expressing the RNA-protein complex
and isolating the bioreactor cells from the other cells in culture
before obtaining target cells from a subject.
[0192] In any of these methods, the subjects of the steps are a
mammalian animal, including a human. In any of the ex vivo methods
described herein, the subject from which the target cells are
obtained and the subject to which the cells are administered is a
mammalian animal subject, including, for example a human subject.
The expression vector can be any of the expression vectors
described herein. The RNA-protein complex can be any of the
RNA-protein complexes described herein. In one embodiment, the
biologically active RNA of the RNA-protein complex is an shRNA. In
another embodiment, the biologically active RNA of the RNA-protein
complex is an aptamer. In one embodiment, the RNA-protein complex
encoded by the expression vector comprises a non-classical
secretory domain sequence. In another embodiment, the RNA-protein
complex encoded by the expression vector comprises a cell
penetrating peptide. In another embodiment, the RNA-protein complex
encoded by the expression vector comprises a cell penetrating
peptide and a non-classical secretory domain. In one embodiment,
the cells of step (b) or step (c) are stably transfected with the
expression vector.
[0193] The invention also provides methods of using the bioreactor
cells in vivo for the delivery of a biologically active RNA to
target cells and/or tissues. In one embodiment, the method of
delivering a biologically active RNA to target cells in vivo
comprises the steps of: (a) preparing an expression vector that
encodes an RNA-protein complex comprising one or more biologically
active RNAs, a recognition RNA sequence, optionally a terminal
minihelix sequence, an RNA binding domain, and one or more
transport peptide sequences (i.e., selected from a cell penetrating
domain, non-classical secretory domain, endosomal release domain,
fusogenic peptide, and a receptor binding domain); (b)
administering the expression vector of step (a) to cells in culture
to produce bioreactor cells expressing the RNA-protein complex; (c)
collecting the cultured cells of step (b); (d) administering the
cells in step (c) to a subject. In one embodiment, the subject of
step (d) is a mammalian animal. In one embodiment, the mammalian
animal is a human subject.
[0194] In certain embodiments of the method, the expression vector
of step (a) further comprises a polynucleotide sequence encoding a
nucleic acid comprising one or more biologically active RNA
sequences that target one or more further gene target(s). In one
embodiment, the additional polynucleotide sequence encodes a
nucleic acid comprising one or more biologically active RNA
sequences that target a further gene target and an RNA recognition
sequence. In another embodiment, where one of the biologically
active RNA sequences in the vector is a short interfering RNA
(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), or short
hairpin RNA (shRNA), the additional polynucleotide sequence encodes
a nucleic acid comprising one or more biologically active RNA
sequences targeted to Dicer and/or Drosha.
[0195] The invention also provides methods of using the bioreactor
cells in vivo for the delivery of a biologically active RNA to
target cells and/or tissues. In one embodiment, the method of
delivering a biologically active RNA to target cells in vivo
comprises the steps of: (a) preparing an expression vector that
encodes an RNA-protein complex comprising one or more biologically
active RNAs, a recognition RNA sequence, optionally a terminal
minihelix sequence, an RNA binding domain, and one or more
transport peptide sequences and one or more polynucleotide
sequences encoding one or more viral polymerases and one or more
viral accessory proteins necessary for viral replication; (b)
preparing an expression vector comprising one or more
polynucleotide sequences encoding one or more viral coat proteins
and one or more viral fusogenic proteins; (c) administering the
expression vector of step (a) and the expression vector of step (b)
to cells in culture to produce bioreactor cells expressing the
RNA-protein complex; (d) collecting the cultured cells of step (c);
(e) administering the cells in step (d) to a subject. In one
embodiment, the subject of step (e) is a mammalian animal. In one
embodiment, the mammalian animal is a human subject.
[0196] In certain embodiments of the method, the expression vector
of step (a) further comprises a polynucleotide sequence encoding a
nucleic acid comprising one or more biologically active RNA
sequences that target one or more further gene target(s). In one
embodiment, the additional polynucleotide sequence encodes a
nucleic acid comprising one or more biologically active RNA
sequences that target a further gene target and an RNA recognition
sequence. In another embodiment, where one of the biologically
active RNA sequences in the vector is a short interfering RNA
(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), or short
hairpin RNA (shRNA), the additional polynucleotide sequence encodes
a nucleic acid comprising one or more biologically active RNA
sequences targeted to Dicer and/or Drosha.
[0197] The invention also provides methods of using the bioreactor
cells in vivo for the delivery of a biologically active RNA to
target cells and/or tissues. In one embodiment, the method of
delivering a biologically active RNA to target cells in vivo
comprises the steps of: (a) preparing an expression vector
comprising a polynucleotide sequence encoding nucleic acid
comprising one or more biologically active RNAs and one or more
polynucleotide sequences encoding one or more viral polymerases and
one or more viral accessory proteins necessary for viral
replication; (b) preparing an expression vector comprising one or
more polynucleotide sequences encoding one or more viral coat
proteins and one or more viral fusogenic proteins; (c)
administering the expression vector of step (a) and the expression
vector of (b) to cells in culture to produce bioreactor cells
expressing the RNA-protein complex; (d) collecting the cultured
cells of step (c); (e) administering the cells in step (d) to a
subject. In one embodiment, the subject of step (e) is a mammalian
animal. In one embodiment, the mammalian animal is a human
subject.
[0198] The invention also provides methods of using the bioreactor
cells in vivo for the delivery of a biologically active RNA to
target cells and/or tissues. In one embodiment, the method of
delivering a biologically active RNA to target cells in vivo
comprises the steps of: (a) preparing an expression vector
comprising a polynucleotide sequence encoding nucleic acid
comprising one or more biologically active RNAs, a recognition RNA
sequence, and optionally a terminal minihelix sequence; (b)
preparing an expression vector comprising a polynucleotide sequence
encoding a polypeptide comprising an RNAs binding domain and one or
more transport peptides; (c) administering the expression vector of
step (a) and the expression vector of (b) to cells in culture to
produce bioreactor cells expressing the RNA-protein complex; (d)
collecting the cultured cells of step (c); (e) administering the
cells in step (d) to a subject. In one embodiment, the subject of
step (e) is a mammalian animal. In one embodiment, the mammalian
animal is a human subject.
[0199] In any of the above described methods, the method can
further comprise the steps of: testing the cells of (c) or (d) to
determine the bioreactor cells expressing the RNA-protein complex
and isolating the bioreactor cells from the other cells in culture
before administering the cells to a subject. In one embodiment, the
subject is a mammalian animal. In one embodiment, the mammalian
animal is a human subject.
[0200] Methods of Treatment
[0201] In one embodiment, the invention provides a method of
preventing, ameliorating, and/or treating a disease or condition
associated with defective gene expression and/or activity in a
subject comprising administering to the subject an expression
vector of the invention. Any of the expression vector described
herein can be used in the methods for preventing, ameliorating,
and/or treating a disease or condition associated with defective
gene expression and/or activity in a subject.
[0202] In one embodiment, the invention provides a method of
preventing, ameliorating, and/or treating a disease or condition
associated with defective gene expression and/or activity in a
subject comprising administering to the subject an expression
vector comprising a polynucleotide encoding a nucleic acid
comprising one or more biologically active RNA sequences directed
to a target gene, a recognition RNA sequence, and optionally a
terminal minihelix sequence and a polynucleotide encoding a
polypeptide comprising an RNA binding domain and one or more
transport peptide sequences (i.e., selected from a cell penetrating
peptide sequence, non-classical secretory domain, endosomal release
domain, and a receptor binding domain). In one embodiment, the
expression vector further comprises a polynucleotide encoding a
further nucleic acid comprising one or more biologically active RNA
sequences directed to a target gene(s), optionally a recognition
RNA binding domain, and optionally a terminal minihelix sequence.
In one embodiment, the target gene(s) of the further nucleic acid
is selected from Dicer and/or Drosha.
[0203] In one embodiment, the invention provides a method of
preventing, ameliorating, and/or treating a disease or condition
associated with defective gene expression and/or activity in a
subject comprising administering to the subject an expression
vector comprising a polynucleotide encoding a nucleic acid
comprising one or more biologically active RNA sequences directed
to a target gene, a recognition RNA sequence, and optionally a
terminal minihelix sequence and a polynucleotide encoding a
polypeptide comprising an RNA binding domain and one or more
transport peptide sequences and one or more polynucleotide
sequences encoding one or more viral polymerases and one or more
viral accessory proteins necessary for viral replication and an
expression vector comprising one or more polynucleotide sequences
encoding one or more viral coat proteins and one or more viral
fusogenic proteins. In one embodiment, the expression vector
further comprises a polynucleotide encoding a further nucleic acid
comprising one or more biologically active RNA sequences directed
to a target gene(s), optionally a recognition RNA binding domain,
and optionally a terminal minihelix sequence. In one embodiment,
the target gene(s) of the further nucleic acid is selected from
Dicer and/or Drosha.
[0204] In one embodiment, the invention provides a method of
preventing, ameliorating, and/or treating a disease or condition
associated with defective gene expression and/or activity in a
subject comprising administering to the subject an expression
vector comprising a polynucleotide encoding a nucleic acid
comprising one or more biologically active RNA sequences directed
to a target gene and one or more polynucleotide sequences encoding
one or more viral polymerases and one or more viral accessory
proteins necessary for viral replication and an expression vector
comprising one or more polynucleotide sequences encoding one or
more viral coat proteins and one or more viral fusogenic
proteins;
[0205] In one embodiment, the invention provides a method of
preventing, ameliorating, and/or treating a disease or condition
associated with defective gene expression and/or activity in a
subject comprising administering to the subject a first expression
vector encoding a nucleic acid comprising one or more biologically
active RNA sequences directed to a target gene, a recognition RNA
sequence, and optionally a terminal minihelix sequence and a second
expression vector encoding a polypeptide comprising an RNA binding
domain and one or more transport peptide sequences (i.e, selected
from a cell penetrating peptide sequence, non-classical secretory
domain, endosomal release domain, and a receptor binding domain).
In one embodiment, the method further comprises administering to
the subject a third expression vector encoding a nucleic acid
comprising one or more biologically active RNA sequences directed
to a target gene(s), optionally a recognition RNA binding domain,
and optionally a terminal minihelix sequence. In one embodiment,
the target gene(s) of the second nucleic acid is selected from
Dicer and/or Drosha.
[0206] In any of the above-described methods, the expression
vectors can be administered as a composition comprising the
expression vectors and a pharmaceutically acceptable carrier.
[0207] The invention additionally provides a method of preventing,
ameliorating, and/or treating a disease or condition associated
with defective gene expression and/or activity in a subject
comprising administering to the subject one or more bioreactor
cells of the invention. In one embodiment, the invention provides a
method of preventing, ameliorating, and/or treating a disease or
condition associated with defective gene expression and/or activity
in a subject comprising administering to the subject a composition
comprising one or more bioreactor cells of the invention and a
pharmaceutically acceptable carrier including but not limited to
phosphate buffered saline, saline or 5% dextrose. The bioreactor
cell(s) can be any of the bioreactor cell(s) of the invention
described herein. In one embodiment, the bioreactor cell encodes an
RNA-protein complex comprising one or more biologically active RNA
sequences directed to a target gene, a recognition RNA sequence,
optionally a terminal minihelix sequence, an RNA binding domain
sequence, and one or more transport peptide sequences selected from
a cell penetrating peptide sequence, non-classical secretory
domain, endosomal release domain, receptor binding domain, and
fusogenic peptide.
[0208] In another embodiment, the invention provides a method of
preventing, ameliorating, and/or treating a disease or condition
associated with defective gene expression and/or activity in a
subject comprising administering to the subject a composition
comprising one or more bioreactor cells and a pharmaceutically
acceptable carrier including but not limited to phosphate buffered
saline, saline or 5% dextrose, wherein the bioreactor cell(s)
produces and secretes an RNA-protein complex comprising one or more
biologically active RNA sequences directed to a target gene(s), a
recognition RNA sequence, and optionally a terminal minihelix
sequence, an RNA binding domain sequence, one or more transport
peptide sequences selected from a cell penetrating peptide
sequence, non-classical secretory domain, endosomal release domain,
receptor binding domain, and further produces an RNA comprising one
or more biologically active RNA sequences directed to Dicer and/or
Drosha.
[0209] In any of the above described methods of preventing,
ameliorating, and/or treating a disease or condition associated
with defective gene expression and/or activity, suitable gene
targets include Mmp2, Vascular Endothelial Growth Factor (VEGF),
Vascular Endothelial Growth Factor Receptor (VEGFR), Cav-1,
Epidermal Growth Factor Receptor (EGFR), H-Ras,Bcl-2, Survivin,
FAK, STAT-3, HER-3, Beta-Catenin, and Src.
[0210] Thus, in one embodiment, the present invention provides a
method of preventing, ameliorating, and/or treating a disease or
condition associated with defective target gene expression and/or
activity in a subject comprising administering to the subject a
composition comprising one or more expression vectors and a
pharmaceutically acceptable carrier, wherein the expression
vector(s) encodes an RNA-protein complex comprising one or more
biologically active RNA sequences directed to the target gene, a
recognition RNA sequence, optionally a terminal minihelix sequence,
an RNA binding domain sequence, and one or more sequences selected
from a cell penetrating peptide sequence, non-classical secretory
domain, endosomal release domain, receptor binding domain, and
fusogenic peptide. Exemplary target genes include Mmp2, Vascular
Endothelial Growth Factor (VEGF), Vascular Endothelial Growth
Factor Receptor (VEGFR), Cav-1, Epidermal Growth Factor Receptor
(EGFR), H-Ras,Bcl-2, Survivin, FAX, STAT-3, HER-3, Beta-Catenin,
and Src.
[0211] In another embodiment, the present invention provides a
method of preventing, ameliorating, and/or treating a disease or
condition associated with defective gene expression and/or activity
in a subject comprising administering to the subject a composition
comprising one or more bioreactor cells and a pharmaceutically
acceptable carrier, wherein the defective gene expression and/or
activity is selected from defective Mmp2, Vascular Endothelial
Growth Factor (VEGF), Vascular Endothelial Growth Factor Receptor
(VEGFR), Cav-1, Epidermal Growth Factor Receptor (EGFR),
H-Ras,Bcl-2, Survivin, FAK, STAT-3, HER-3, Beta-Catenin, and Src
expression and/or activity and wherein the bioreactor cell(s)
produces and secretes an RNA-protein complex comprising one or more
biologically active RNA sequences, a recognition RNA sequence,
optionally a terminal minihelix sequence, an RNA binding domain
sequence, and one or more sequences selected from a cell
penetrating peptide sequence, non-classical secretory domain,
endosomal release domain, receptor binding domain, wherein the
biologically active RNA(s) is directed to a gene(s) selected from
Mmp2, Vascular Endothelial Growth Factor (VEGF), Vascular
Endothelial Growth Factor Receptor (VEGFR), Cav-1, Epidermal Growth
Factor Receptor (EGFR), H-Ras,Bcl-2, Survivin, FAK, STAT-3, HER-3,
Beta-Catenin, and Src and wherein the biologically active RNA(s)
targets the gene(s) having defective expression and/or
activity.
[0212] Polynucleotides and Polypeptides of the Invention
[0213] The present invention provides novel polynucleotides useful
in the production of nucleic acid molecules, polypeptides,
RNA-protein complexes, and expression vectors comprising the same,
for the delivery of biologically active RNAs to cells. In one
embodiment, the invention provides an isolated polynucleotide that
encodes a nucleic acid molecule comprising one or more biologically
active RNA sequences, a recognition RNA sequence, and optionally a
terminal minihelix sequence. In one specific embodiment, the
isolated polynucleotide encodes a nucleic acid molecule comprising
one or more short hairpin RNAs, a recognition RNA sequence, and
optionally a terminal minihelix sequence. In another embodiment,
the isolated polynucleotide encodes a nucleic acid molecule
comprising one or more aptamers, a recognition RNA sequence, and
optionally a terminal minihelix sequence. In another embodiment,
the isolated polynucleotide encodes a nucleic acid molecule
comprising one or more ribozymes, a recognition RNA sequence, and
optionally a terminal minihelix sequence. In another embodiment,
the isolated polynucleotide encodes a nucleic acid molecule
comprising one or more antisense nucleic acids, a recognition RNA
sequence, and optionally a terminal minihelix sequence. In
addition, the invention provides an isolated polynucleotide that
encodes a nucleic acid molecule comprising one or more biologically
active RNA sequences targeted to Dicer, for example, a
polynucleotide comprising SEQ ID NO: 49.
[0214] In addition, the invention provides a novel fusion protein
comprising an amino acid sequence (RNA binding domain) that binds
to the recognition RNA sequence of the above-described nucleic acid
sequence and an amino acid sequence that facilitates the transport
and secretion of the above-described biologically active RNA from a
cell (transport peptide). Thus, in one embodiment, the fusion
protein comprises an RNA binding domain and one or more transport
peptides. The transport peptide of the fusion polypeptide can be
any amino acid sequence that facilitates the delivery of nucleic
acids, peptides, fusion proteins, RNA-protein complexes, and/or
other biological molecules to the extracellular matrix and/or to
neighboring cells and tissues.
[0215] The invention also provides an isolated polynucleotide that
encodes any of the polypeptide molecules described herein. In one
embodiment, the invention provides an isolated polynucleotide that
encodes a polypeptide comprising an amino acid sequence of an RNA
binding domain and a polypeptide comprising an amino acid sequence
of one or more transport peptide sequences, for example, selected
from a non-classical secretory domain, a cell penetrating peptide,
a receptor binding domain, an endosomal release domain, and a
fusogenic peptide.
[0216] In any of the above-described embodiments of the isolated
polynucleotide encoding a nucleic acid or polypeptide of the
invention, the isolated polynucleotide can comprise a sequence
wherein the individual sequences, domains and peptides are joined
directly without the addition of one or more linker, spacer, or
other sequences or are joined with the addition of one or more
linker, spacer, and/or other sequences.
[0217] The invention also provides the complementary sequence of
any of the polynucleotides described in this section and elsewhere
in the application. As used herein, the term "complementary" refers
to the hybridization or base pairing between nucleotides, such as,
for example, between the two strands of a double-stranded
polynucleotide or between an oligonucleotide primer and a primer
binding site on a single-stranded polynucleotide to be amplified or
sequenced. Two single-stranded nucleotide molecules are said to be
complementary when the nucleotides of one strand, optimally aligned
with appropriate nucleotide insertions, deletions or substitutions,
pair with at least about 80% of the nucleotides of the other
strand.
[0218] A "polynucleotide" of the invention also includes those
polynucleotides capable of hybridizing, under stringent
hybridization conditions, to any of the polynucleotides described
herein or the complements thereof. "Stringent hybridization
conditions" are generally selected to be about 5.degree. C. lower
than the thermal melting point (T.sub.M) for the specific sequence
at a defined ionic strength and pH. One example of stringent
hybridization conditions refers to an overnight incubation at
42.degree. C. in a solution comprising 50% formamide, 5.times.SSC
(750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH
7.6), 5.times.Denhardt's solution, 10% dextran sulfate, and 20
.mu.g/ml denatured, sheared salmon sperm DNA, followed by washing
the filters in 0.1.times.SSC at about 65.degree. C.
[0219] The invention also relates to polynucleotides comprising
nucleotide sequences having at least 80% identity over their entire
length with any of the polynucleotides of the invention, for
example, at least 85%, at least 90% identity, at least 95%
identity, at least 98% identity, and at least 99% identity. Thus,
in certain specific embodiments, the invention provides an isolated
polynucleotide comprising nucleotide sequence having at least 80%
identity (i.e., at least 85%, 90%, 95%, 98%, or 99% identity) over
its entire length to a polynucleotide encoding a nucleic acid
molecule comprising one or more sequences selected from SEQ ID NOs:
1-15 and a sequence selected from SEQ ID NOs: 16-23.
[0220] In one embodiment, the invention provides an isolated
polynucleotide comprising a nucleotide sequence having at least 80%
(i.e., at least 85%, 90%, 95%, 98%, or 99% identity) identity over
its entire length to a polynucleotide encoding a polypeptide
comprising an amino acid sequence selected from SEQ ID NOs: 24-31.
In another embodiment, the invention provides an isolated
polynucleotide comprising a nucleotide sequence having at least 80%
identity over its entire length to a polynucleotide encoding a
polypeptide comprising an amino acid sequence selected from SEQ ID
NOs: 24-31 and a sequence selected from SEQ ID NOs: 32-40. In
another embodiment, the invention provides an isolated
polynucleotide comprising a nucleotide sequence having at least 80%
identity over its entire length to a polynucleotide encoding a
polypeptide comprising an amino acid sequence selected from SEQ ID
NOs: 50-54. In another embodiment, the invention provides an
isolated polynucleotide comprising a nucleotide sequence having at
least 80% identity over its entire length to a polynucleotide
encoding a polypeptide comprising an amino acid sequence selected
from SEQ ID NOs: 24-31 and a sequence selected from SEQ ID NOs:
41-48. In another embodiment, the invention provides an isolated
polynucleotide comprising a nucleotide sequence having at least 80%
identity over its entire length to a polynucleotide encoding a
polypeptide comprising an amino acid sequence selected from SEQ ID
NOs: 24-31, a sequence selected from SEQ ID NOs: 32-40, and a
sequence selected from SEQ ID NOs: 41-48.
[0221] The invention also relates to polynucleotide and polypeptide
variants. "Polynucleotide variant" refers to a polynucleotide
differing from the polynucleotide of the invention, but retaining
essential properties thereof. Likewise, "polypeptide variant"
refers to a polypeptide differing from the polypeptide of the
present invention, but retaining essential properties thereof. In
certain embodiments, the invention provides a polynucleotide
variant of a sequence selected from SEQ ID NOs: 1-23. In certain
embodiments, the invention provides a polynucleotide encoding a
polypeptide variant of a sequence selected from SEQ ID NOs:
24-54.
[0222] Variants include, but are not limited to, splice variants
and allelic variants, as well as addition, deletion, truncation,
and substitution variants. "Allelic variants" are
naturally-occurring variants that refer to one of several alternate
forms of a gene occupying a given locus on a chromosome of an
organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New
York (1985).) These allelic variants can vary at either the
polynucleotide and/or polypeptide level. Alternatively,
non-naturally occurring variants may be produced by mutagenesis
techniques or by direct synthesis.
[0223] Variants can include sequences having "conservative amino
acid substitution", which term refers to a substitution of a native
amino acid residue with a normative residue such that there is
little or no effect on the polarity or charge of the amino acid
residue at that position. For example, a conservative substitution
results from the replacement of a non-polar residue in a
polypeptide with any other non-polar residue. Another example of a
conservative substitution is the replacement of an acidic residue
with another acidic residue. Variants can also include "orthologs",
which term refers to a polypeptide that corresponds to a
polypeptide identified from a different species.
[0224] In a particular embodiment, the transport polypeptide
comprises one or more substitutions, deletions, truncations,
additions and/or insertions, such that the bioactivity of the
native transport polypeptide is not substantially diminished. In
other words, the bioactivity of a transport polypeptide variant may
be diminished by, less than 50%, and preferably less than 20%,
relative to the native protein.
[0225] Preferably, a transport polypeptide variant contains
conservative substitutions. A "conservative substitution" is one in
which an amino acid is substituted for another amino acid that has
similar properties, such that one skilled in the art of peptide
chemistry would expect the secondary structure and hydropathic
nature of the polypeptide to be substantially unchanged. Amino acid
substitutions may generally be made on the basis of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity and/or
the amphipathic nature of the residues. For example, negatively
charged amino acids include aspartic acid and glutamic acid;
positively charged amino acids include lysine and arginine; and
amino acids with uncharged polar head groups having similar
hydrophilicity values include leucine, isoleucine and valine;
glycine and alanine; asparagine and glutamine; and serine,
threonine, phenylalanine and tyrosine. A variant may also, or
alternatively, contain nonconservative changes. In a particular
embodiment, variant polypeptides differ from a native sequence by
substitution, deletion or addition of amino acids. Variants may
also (or alternatively) be modified by, for example, the deletion
or addition of amino acids that have minimal influence on the
bioactivity, secondary structure and hydropathic nature of the
polypeptide.
[0226] The invention provides methods for isolating or recovering a
nucleic acid encoding a polypeptide having a transport polypeptide
activity from a biological sample comprising the steps of: (a)
providing an amplification primer sequence pair for amplifying a
nucleic acid encoding a polypeptide of interest, wherein the primer
pair is capable of amplifying a nucleic acid of the invention; (b)
isolating a nucleic acid from the biological sample or treating the
biological sample such that nucleic acid in the sample is
accessible for hybridization to the amplification primer pair; and,
(c) combining the nucleic acid of step (b) with the amplification
primer pair of step (a) and amplifying nucleic acid from the
biological sample, thereby isolating or recovering a nucleic acid
encoding a polypeptide having a transport polypeptide activity from
a biological sample. One or each member of the amplification primer
sequence pair can comprise an oligonucleotide comprising at least
about 10 to 50 consecutive bases of a sequence of the invention. In
one aspect, the biological sample can be derived from a bacterial
cell, a protozoan cell, an insect cell, a yeast cell, a plant cell,
a fungal cell or a mammalian cell.
[0227] The invention provides methods of generating a variant of a
nucleic acid encoding a transport polypeptide having a transport
polypeptide activity comprising the steps of: (a) providing a
template nucleic acid comprising a nucleic acid of the invention;
and (b) modifying, deleting or adding one or more nucleotides in
the template sequence, or a combination thereof, to generate a
variant of the template nucleic acid. In one aspect, the method can
further comprise expressing the variant nucleic acid to generate a
variant transport polypeptide polypeptide. The modifications,
additions or deletions can be introduced by a method comprising
error-prone PCR, shuffling, oligonucleotide-directed mutagenesis,
assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette
mutagenesis, recursive ensemble mutagenesis, exponential ensemble
mutagenesis, site-specific mutagenesis, gene reassembly, gene site
saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR)
or a combination thereof. In another aspect, the modifications,
additions or deletions are introduced by a method comprising
recombination, recursive sequence recombination,
phosphothioate-modified DNA mutagenesis, uracil-containing template
mutagenesis, gapped duplex mutagenesis, point mismatch repair
mutagenesis, repair-deficient host strain mutagenesis, chemical
mutagenesis, radiogenic mutagenesis, deletion mutagenesis,
restriction-selection mutagenesis, restriction-purification
mutagenesis, artificial gene synthesis, ensemble mutagenesis,
chimeric nucleic acid multimer creation and a combination
thereof.
[0228] In one aspect, the method can be iteratively repeated until
a transport polypeptide having an altered or different activity or
an altered or different stability from that of a polypeptide
encoded by the template nucleic acid is produced. In one aspect,
the method can be iteratively repeated until a transport protein
coding sequence having an altered codon usage from that of the
template nucleic acid is produced. In another aspect, the method
can be iteratively repeated until a transport protein having higher
or lower level of message expression or stability from that of the
template nucleic acid is produced.
[0229] The invention provides methods for modifying codons in a
nucleic acid encoding a polypeptide having transport protein
activity to increase its expression in a host cell, the method
comprising the following steps: (a) providing a nucleic acid of the
invention encoding a polypeptide having transport protein activity;
and, (b) identifying a non-preferred or a less preferred codon in
the nucleic acid of step (a) and replacing it with a preferred or
neutrally used codon encoding the same amino acid as the replaced
codon, wherein a preferred codon is a codon over-represented in
coding sequences in genes in the host cell and a non-preferred or
less preferred codon is a codon under-represented in coding
sequences in genes in the host cell, thereby modifying the nucleic
acid to increase its expression in about cell.
[0230] The invention provides methods for modifying codons in a
nucleic acid encoding a polypeptide having transport protein
activity; the method comprising the following steps: (a) providing
a nucleic acid of the invention; and, (b) identifying a codon in
the nucleic acid of step (a) and replacing it with a different
codon encoding the same amino acid as the replaced codon, thereby
modifying codons in a nucleic acid encoding a transport
protein.
[0231] The invention provides methods for modifying codons in a
nucleic acid encoding a polypeptide having transport protein
activity to increase its expression in a host cell, the method
comprising the following steps: (a) providing a nucleic acid of the
invention encoding a transport protein polypeptide; and, (b)
identifying a non-preferred or a less preferred codon in the
nucleic acid of step (a) and replacing it with a preferred or
neutrally used codon encoding the same amino acid as the replaced
codon, wherein a preferred codon is a codon over-represented in
coding sequences in genes in the host cell and a non-preferred or
less preferred codon is a codon under-represented in coding
sequences in genes in the host cell, thereby modifying the nucleic
acid to increase its expression in a host cell.
[0232] The invention provides methods for modifying a codon in a
nucleic acid encoding a polypeptide having a transport protein
activity to decrease its expression in a host cell, the method
comprising the following steps: (a) providing a nucleic acid of the
invention; and (b) identifying at least one preferred codon in the
nucleic acid of step (a) and replacing it with a non-preferred or
less preferred codon encoding the same amino acid as the replaced
codon, wherein a preferred codon is a codon over-represented in
coding sequences in genes in a host cell and a non-preferred or
less preferred codon is a codon under-represented in coding
sequences in genes in the host cell, thereby modifying the nucleic
acid to decrease its expression in a host cell. In one aspect, the
host cell can be a bacterial cell, a fungal cell, an insect cell, a
yeast cell, a plant cell or a mammalian cell.
[0233] The invention provides methods for producing a library of
nucleic acids encoding a plurality of modified transport protein
active sites or substrate binding sites, wherein the modified
active sites or substrate binding sites are derived from a first
nucleic acid comprising a sequence encoding a first active site or
a first substrate binding site the method comprising the following
steps: (a) providing a first nucleic acid encoding a first active
site or first substrate binding site, wherein the first nucleic
acid sequence comprises a sequence that hybridizes under stringent
conditions to a nucleic acid of the invention, and the nucleic acid
encodes a transport protein active site or a transport protein
substrate binding site; (b) providing a set of mutagenic
oligonucleotides that encode naturally-occurring amino acid
variants at a plurality of targeted codons in the first nucleic
acid; and, (c) using the set of mutagenic oligonucleotides to
generate a set of active site-encoding or substrate binding
site-encoding variant nucleic acids encoding a range of amino acid
variations at each amino acid codon that was mutagenized, thereby
producing a library of nucleic acids encoding a plurality of
modified transport protein active sites or substrate binding sites.
In one aspect, the method comprises mutagenizing the first nucleic
acid of step (a) by a method comprising an optimized directed
evolution system, gene site-saturation mutagenesis (GSSM),
synthetic ligation reassembly (SLR), error-prone PCR, shuffling,
oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR
mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive
ensemble mutagenesis, exponential ensemble mutagenesis,
site-specific mutagenesis, gene reassembly, gene site saturated
mutagenesis (GSSM), synthetic ligation reassembly (SLR) and a
combination thereof. In another aspect, the method comprises
mutagenizing the first nucleic acid of step (a) or variants by a
method comprising recombination, recursive sequence recombination,
phosphothioate-modified DNA mutagenesis, uracil-containing template
mutagenesis, gapped duplex mutagenesis, point mismatch repair
mutagenesis, repair-deficient host strain mutagenesis, chemical
mutagenesis, radiogenic mutagenesis, deletion mutagenesis,
restriction-selection mutagenesis, restriction-purification
mutagenesis, artificial gene synthesis, ensemble mutagenesis,
chimeric nucleic acid multimer creation and a combination
thereof.
[0234] Thus, the invention includes those polynucleotides that
encode a nucleic acid or polypeptide of the invention, including
the described substitution, deletion, truncation, and insertion
variants, as well as allelic variants, splice variants, fragments,
derivatives, and orthologs. Accordingly, the polynucleotide
sequences of the invention include both the naturally occurring
sequences as well as variant forms. Likewise, the polypeptides of
the invention encompass both naturally occurring proteins as well
as variations and modified forms thereof. Such variants will
continue to possess the desired activity. The deletions,
insertions, and substitutions of the polypeptide sequence
encompassed herein are not expected to produce radical changes in
the characteristics of the polypeptide. However, when it is
difficult to predict the exact effect of the substitution,
deletion, or insertion in advance of doing so, one skilled in the
art will appreciate that the effect will be evaluated by routine
screening assays.
[0235] Administration of Expression Vectors
[0236] The expression vectors of the invention are administered to
cells and/or mammalian subjects so as to modulate target gene
expression, for example, in the treatment, prevention, and/or
amelioration of a disorder associated with defective target gene
expression and/or activity.
[0237] The expression vectors of the invention and formulations
thereof can be delivered by local or systemic administration and
can be administered by a variety of routes including orally,
topically, rectally or via parenteral, intranasal, intradermal,
intra-arterial, intravenous and intramuscular routes, as well as by
direct injection into diseased tissue. The term parenteral is meant
to include percutaneous, subcutaneous, intravascular,
intramuscular, as well as intrathecal injection or infusion
techniques and the like. The expression vector can be directly
injected into the brain. Alternatively, the vector can be
introduced intrathecally for brain and spinal cord conditions. In
another example, the vector can be introduced intramuscularly.
Direct injection of the vectors of the invention, whether
subcutaneous, intramuscular, or intradermal, can take place using
standard needle and syringe methodologies, or by known needle-free
technologies. Traditional approaches to CNS delivery are known and
include, for example, intrathecal and intracerebroventricular
administration, implantation of catheters and pumps, direct
injection or perfusion at the site of injury or lesion, injection
into the brain arterial system, or by chemical or osmotic opening
of the blood-brain barrier. The vectors of the invention and
formulations thereof can be administered via pulmonary delivery,
such as by inhalation of an aerosol or spray dried formulation
administered by an inhalation device or nebulizer, providing rapid
local uptake of the vectors into relevant pulmonary tissues. The
compositions of the invention can also be formulated and used as
creams, gels, sprays, oils and other suitable compositions for
topical, dermal, or transdermal administration as is known in the
art.
[0238] Dosing frequency will depend upon the pharmacokinetic
parameters of the expression vector in the formulation used.
Typically, a clinician administers the composition until a dosage
is reached that achieves the desired effect. The composition can
therefore be administered as a single dose, or as two or more doses
(which may or may not contain the same amount of the desired
vector) over time, or as a continuous infusion via an implantation
device or catheter. Further refinement of the appropriate dosage is
routinely made by those of ordinary skill in the art and is within
the ambit of tasks routinely performed by them. Thus,
administration of the expression vectors in accordance with the
present invention is effected in one dose or can be administered
continuously or intermittently throughout the course of treatment,
depending, for example, upon the recipient's physiological
condition, whether the purpose of the administration is therapeutic
or prophylactic, and other factors known to skilled practitioners.
The administration of the expression vectors of the invention can
be essentially continuous over a preselected period of time or can
be in a series of spaced doses.
[0239] An effective amount of vector to be added can be empirically
determined. Methods of determining the most effective means and
dosages of administration are well known to those of skill in the
art and will vary with the vector, the target cells, and the
subject being treated. For example, the amount to be administered
depends on several factors including, but not limited to, the
RNA-protein complex, the disorder, the weight, physical condition,
and the age of the mammal, and whether prevention or treatment is
to be achieved. Such factors can be readily determined by the
clinician employing animal models or other test systems which are
well known in the art. For example, appropriate dosages may be
ascertained through use of appropriate dose-response data. Thus,
single and multiple administrations can be carried out with the
dose level and pattern being selected by the treating physician. A
pharmaceutically effective dose is that dose required to prevent,
inhibit the occurrence, or treat (alleviate a symptom) of a disease
state. In general, as mentioned, a pharmaceutically effective dose
depends on the type of disease, the composition used, the route of
administration, the type of mammal being treated, the physical
characteristics of the specific mammal under consideration,
concurrent medication, and other factors that those skilled in the
medical arts will recognize. Generally, an amount between 0.1 mg/kg
and 100 mg/kg body weight/day of active ingredients is
administered.
[0240] It also may be desirable to use pharmaceutical compositions
of the vectors according to the invention ex vivo. In such
instances, cells, tissues or organs that have been removed from the
subject are exposed to vectors pharmaceutical compositions after
which the cells, tissues and/or organs are subsequently implanted
back into the subject.
[0241] Pharmaceutical Compositions
[0242] The invention provides a pharmaceutical composition
comprising one or more expression vectors of the invention in an
acceptable carrier, such as a stabilizer, buffer, solubilizer,
emulsifier, preservative and/or adjuvant. Preferably, acceptable
formulation materials are nontoxic to recipients at the dosages and
concentrations employed. The vectors of the invention can be
administered to a subject by any standard means, with or without
stabilizers, buffers, and the like, to form a pharmaceutical
composition. A pharmacological composition or formulation refers to
a composition or formulation that allows for the effective
distribution of the vectors of the instant invention in a form
suitable for administration, e.g., systemic or local
administration, into a cell or subject, including for example a
human. Suitable forms, in part, depend upon the use or the route of
entry, for example oral, transdermal, or by injection. Such forms
should be administered in the physical location most suitable for
the desired activity and should not prevent the composition or
formulation from reaching a target cell. In one embodiment, the
pharmaceutical composition comprises sufficient vector to produce a
therapeutically effective amount of the RNA-protein complex, i.e.,
an amount sufficient to reduce or ameliorate symptoms of the
disease state in question or an amount sufficient to confer the
desired benefit. The pharmaceutical compositions can also contain a
pharmaceutically acceptable excipient, for example, sorbitol,
Tween80, and liquids such as water, saline, glycerol and ethanol.
Pharmaceutically acceptable salts can be included therein, for
example, mineral acid salts such as hydrochlorides, hydrobromides,
phosphates, sulfates, and the like; and the salts of organic acids
such as acetates, propionates, malonates, benzoates, and the like.
Additionally, auxiliary substances, such as wetting or emulsifying
agents, pH buffering substances, and the like, may be present in
such vehicles.
[0243] In certain embodiments, the pharmaceutical composition may
contain formulation materials for modifying, maintaining or
preserving, for example, the pH, osmolarity, viscosity, clarity,
color, isotonicity, odor, sterility, stability, rate of dissolution
or release, adsorption or penetration of the composition. In such
embodiments, suitable formulation materials include, but are not
limited to, amino acids (such as glycine, glutamine, asparagine,
arginine or lysine); antimicrobials; antioxidants (such as ascorbic
acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as
borate, bicarbonate, tris-hcl, citrates, phosphates or other
organic acids); bulking agents (such as mannitol or glycine);
chelating agents (such as ethylenediamine tetraacetic acid (edta));
complexing agents (such as caffeine, polyvinylpyrrolidone,
beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers;
monosaccharides; disaccharides; and other carbohydrates (such as
glucose, mannose or dextrins); proteins (such as serum albumin,
gelatin or immunoglobulins); coloring, flavoring and diluting
agents; emulsifying agents; hydrophilic polymers (such as
polyvinylpyrrolidone); low molecular weight polypeptides;
salt-forming counterions (such as sodium); preservatives (such as
benzalkonium chloride, benzoic acid, salicylic acid, thimerosal,
phenethyl alcohol, methylparaben, propylparaben, chlorhexidine,
sorbic acid or hydrogen peroxide); solvents (such as glycerin,
propylene glycol or polyethylene glycol); sugar alcohols (such as
mannitol or sorbitol); suspending agents; surfactants or wetting
agents (such as pluronics, peg, sorbitan esters, polysorbates such
as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin,
cholesterol, tyloxapal); stability enhancing agents (such as
sucrose or sorbitol); tonicity enhancing agents (such as alkali
metal halides, preferably sodium or potassium chloride, mannitol
sorbitol); delivery vehicles; diluents; excipients and/or
pharmaceutical adjuvants. See REMINGTON'S PHARMACEUTICAL SCIENCES,
18.sup.th edition, (A. R. Gennaro, ed.), 1990, Mack Publishing
Company.
[0244] The expression vectors of the invention and formulations
thereof can be administered orally, topically, parenterally, by
inhalation or spray, or rectally in dosage unit formulations
containing conventional non-toxic pharmaceutically acceptable
carriers, adjuvants and/or vehicles. Compositions intended for oral
use can be prepared according to any method known to the art for
the manufacture of pharmaceutical compositions and such
compositions can contain one or more such sweetening agents,
flavoring agents, coloring agents or preservative agents in order
to provide palatable preparations.
[0245] Aqueous suspensions contain the active materials in a
mixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients include, for example, suspending
agents, for example sodium carboxymethylcellulose, methylcellulose,
hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone,
gum tragacanth and gum acacia; dispersing or wetting agents can be
a naturally-occurring phosphatide, for example, lecithin, or
condensation products of an alkylene oxide with fatty acids, for
example polyoxyethylene stearate, or condensation products of
ethylene oxide with long chain aliphatic alcohols, for example
heptadecaethyleneoxycetanol, or condensation products of ethylene
oxide with partial esters derived from fatty acids and a hexitol
such as polyoxyethylene sorbitol monooleate, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and hexitol anhydrides, for example polyethylene sorbitan
monooleate. The aqueous suspensions can also contain one or more
preservatives, for example ethyl, or n-propyl p-hydroxybenzoate,
one or more coloring agents, one or more flavoring agents, and one
or more sweetening agents, such as sucrose or saccharin.
[0246] Syrups and elixirs can be formulated with sweetening agents,
for example glycerol, propylene glycol, sorbitol, glucose or
sucrose. Such formulations can also contain a demulcent, a
preservative and flavoring and coloring agents. The pharmaceutical
compositions can be in the form of a sterile injectable aqueous or
oleaginous suspension. This suspension can be fog ululated
according to the known art using those suitable dispersing or
wetting agents and suspending agents that have been mentioned
above. The sterile injectable preparation can also be a sterile
injectable solution or suspension in a non-toxic parentally
acceptable diluent or solvent, for example as a solution in
1,3-butanediol. Among the acceptable vehicles and solvents that can
be employed are water, Ringer's solution and isotonic sodium
chloride solution. In addition, sterile, fixed oils are
conventionally employed as a solvent or suspending medium. For this
purpose, any bland fixed oil can be employed including synthetic
mono- or diglycerides. In addition, fatty acids such as oleic acid
find use in the preparation of injectables.
[0247] Methods of Modulating Gene Expression
[0248] The expression vectors of the invention and the Bioreactors
of the invention can be used in vitro, ex vivo, and in vivo to
modulate the expression of a target gene of interest. The invention
provides an expression vector designed to produce an RNA-protein
complex comprising at least one biologically active RNA molecule
targeting one or more genes of interest and a fusion protein
capable of delivering the biologically active RNA molecule(s) to
the extracellular matrix and/or neighboring cells and tissues. The
administration of the expression vector to cells in vivo, ex vivo,
and in vitro converts the cells into "bioreactors" that produce and
deliver biologically active RNA molecules, secreted as RNA-protein
complexes, to the extracellular matrix and/or other neighboring
cells.
[0249] The invention provides methods for modulating the expression
of one or more target gene(s) in a subject comprising administering
to the subject one or more expression vectors of the invention or a
composition(s) thereof. In one embodiment, the method for
modulating the expression of one or more target gene(s) in a
subject comprises administering to the subject an expression vector
comprising a polynucleotide encoding a nucleic acid comprising a
biologically active RNA sequence, recognition RNA sequence,
optionally a terminal minihelix sequence, and a polynucleotide
encoding a polypeptide comprising an RNA binding domain and one or
more transport peptide (i.e., sequences selected from a cell
penetrating peptide sequence, non-classical secretory domain,
endosomal release domain, receptor binding domain, and fusogenic
peptide). In one embodiment, the expression vector comprises a
further nucleic acid comprising one or more biologically active RNA
sequences directed to a target gene(s), optionally a recognition
RNA binding domain, and optionally a terminal minihelix sequence,
wherein the target gene(s) of the further nucleic acid is different
from the target gene of the first nucleic acid. In one embodiment,
the target gene is selected from Dicer and/or Drosha.
[0250] In one embodiment, the method for modulating the expression
of one or more target gene(s) in a subject comprises administering
to the subject an expression vector comprising a polynucleotide
sequence encoding a nucleic acid comprising one or more
biologically active RNA sequences, a recognition RNA sequence, and
optionally a terminal minihelix sequence, a polynucleotide encoding
a polypeptide comprising an RNA binding domain and one or more
transport peptide and one or more polynucleotide sequences encoding
one or more viral polymerases and one or more viral accessory
proteins necessary for viral replication and an expression vector
comprising one or more polynucleotide sequences encoding one or
more viral coat proteins and one or more viral fusogenic proteins.
In one embodiment, the expression vector comprises a further
nucleic acid comprising one or more biologically active RNA
sequences directed to a target gene(s), optionally a recognition
RNA binding domain, and optionally a terminal minihelix sequence,
wherein the target gene(s) of the further nucleic acid is different
from the target gene of the first nucleic acid. In one embodiment,
the target gene is selected from Dicer and/or Drosha.
[0251] In one embodiment, the method for modulating the expression
of one or more target gene(s) in a subject comprises administering
to the subject an expression vector comprising a polynucleotide
sequence encoding a nucleic acid comprising one or more
biologically active RNA sequences and one or more polynucleotide
sequences encoding one or more viral polymerases and one or more
viral accessory proteins necessary for viral replication and an
expression vector comprising one or more polynucleotide sequences
encoding one or more viral coat proteins and one or more viral
fusogenic proteins.
[0252] In another embodiment, the method for modulating the
expression of one or more target gene(s) in a subject comprises
administering to the subject a first expression vector encoding a
nucleic acid comprising one or more biologically active RNA
sequences directed to a target gene, a recognition RNA sequence,
and optionally a terminal minihelix sequence and a second
expression vector encoding a polypeptide comprising an RNA binding
domain and one or more transport peptide sequences (i.e., selected
from a cell penetrating peptide sequence, non-classical secretory
domain, endosomal release domain, receptor binding domain, and
fusogenic peptide) or a composition(s) comprising both expression
vectors. The method can further comprise administering to the
subject a further expression vector encoding a nucleic acid
comprising one or more biologically active RNA sequences directed
to a target gene(s), optionally a recognition RNA binding domain,
and optionally a terminal minihelix sequence, wherein the target
gene(s) is selected from Dicer and/or Drosha.
[0253] The invention also provides a method for modulating the
expression of one or more target gene(s) in a subject comprising
administering to the subject one or more bioreactor cells of the
invention, or a composition thereof, wherein the bioreactor cell(s)
produces and secretes an RNA-protein complex comprising one or more
biologically active RNA sequences directed to a target gene(s), a
recognition RNA sequence, and optionally a terminal minihelix
sequence, an RNA binding domain sequence, one or more transport
peptide (i.e., sequences selected from a cell penetrating peptide
sequence, non-classical secretory domain, endosomal release domain,
receptor binding domain, and fusogenic peptide).
[0254] The subject can be a mammalian subject, including, for
example, a human, rodent, murine, bovine, canine, feline, sheep,
equine, and simian subject. The biologically active RNA sequence
can be a ribozyme, antisense nucleic acid, allozyme, aptamer, short
interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA
(miRNA), short hairpin RNA (shRNA), and a transcript encoding one
or more biologically active peptides; the recognition RNA sequence
can be a U1 loop, Group II intron, NRE stem loop, S1A stem loop,
Bacteriophage Box BR, HIV Rev response element, AMVCP recognition
sequence, and ARE sequence; the RNA binding domain can be a U1A,
CRS1, CRM1, Nucleolin RBD12, hRBMY, Bacteriophage Protein N, HIV
Rev, AMVCP, and tristetrapolin sequence; the cell penetrating
peptide can be a penetratin, transportan, MAP, HIV TAT, Antp, Rev,
FHV coat protein, TP10, and pVEC sequence; and the non-classical
secretory domain can be a Galcetin-1 peptide, Galectin-3 peptide,
IL-1.alpha., IL-1.beta., HASPB, HMGB1, FGF-1, FGF-2, IL-2 signal,
secretory transglutaminase, annexin-1, HIV TAT, Herpes VP22,
thioredoxin, Rhodanese, and plasminogen activator signal nucleotide
sequence. The bioreactor cell can be any of the bioreactor cells
described herein.
[0255] The methods can be used to prevent, ameliorate, and/or treat
a disease or condition associated with defective gene expression
and/or activity in a subject. Suitable gene targets include, for
example, Mmp2, Vascular Endothelial Growth Factor (VEGF), Vascular
Endothelial Growth Factor Receptor (VEGFR), Cav-1, Epidermal Growth
Factor Receptor (EGFR), H-Ras,Bcl-2, Survivin, FAK, STAT-3, HER-3,
Beta-Catenin, and Src. The disorders associated with the defective
expression of these genes are listed in Table V.
[0256] The invention also provides methods for modulating the
expression of a target gene in a target cell ex vivo. In one
embodiment, the invention provides a method for modulating the
expression of a target gene in a target cell ex vivo comprising
administering to the target cell ex vivo one or more expression
vectors of the invention or a composition(s) thereof. In one
specific embodiment, the method comprises the steps of: (a)
obtaining target cells from a subject; (b) administering a
composition comprising one or more expression vector(s) of the
invention and a pharmaceutically acceptable carrier to the target
cells of step (a), wherein the expression vector(s) encodes an
RNA-protein complex of the invention; and (c) administering the
cells in step (b) to said subject. In another embodiment, the
invention provides a method for modulating the expression of a
target gene in a target cell ex vivo comprising administering to
the target cell ex vivo one or more bioreactor cells of the
invention, or a composition thereof, wherein the method comprises
the steps of: (a) obtaining target cells from a subject; (b)
administering a one or more bioreactor cell(s) of the invention to
the target cells of step (a), wherein the bioreactor cell(s)
produces and secretes an RNA-protein complex of the invention; and
(c) administering the cells in step (b) to said subject.
[0257] The invention also provides methods for modulating gene
expression in a cell in culture comprising administering to the
cell one or more expression vectors of the invention or a
composition(s) thereof. Additionally, the invention provides a
method for modulating the expression of one or more target gene(s)
in a cell in culture comprising administering to the cell one or
more bioreactor cells of the invention or a composition
thereof.
[0258] Mechanism of Action for Viral Based Delivery Systems
[0259] The viral based RNA delivery system utilizes an engineered,
replication competent or replication defective virus to deliver
biologically active RNAs from transformed packaging cells to target
cells. This system takes advantage of the capacity virus particles
have to effectively deliver nucleic acids to the interior of target
cells in vitro (Lund P E, et al., Pharm Res. 2009 Dec. 9; Koerber J
T, et al., Mol. Ther. 2008 October; 16(10):1703-9; Cascante A, Gene
Ther. 2007 October; 14(20):1471-80; Ring C J. J Gen Virol. 2002
March; 83(Pt 3):491-502; Parada C, et al., Cancer Gene-Ther. 2003
February; 10(2):152-60; Tiede A, et al., Gene Ther. 2003 October;
10(22):1917-25; Lee Y J, Cancer Gene Ther. 2001 June; 8(6):397-404;
Nestler U, et al., Gene Ther. 1997 November; 4(11):1270-7) and in
vivo (Tseng J C, et al. Gene Ther. 2009 February; 16(2):291-6;
Kikuchi E, et al., Clin Cancer Res. 2007 Aug. 1; 13(15 Pt
1):4511-8; Bourbeau D, et al., Cancer Res. 2007 Apr. 1;
67(7):3387-95; Hiraoka K, et al., Cancer Res. 2007 Jun. 1;
67(11):5345-53; Hiraoka K, et al., Clin Cancer Res. 2006 Dec. 1;
12(23):7108-16; Varghese S, et al., Cancer Res. 2007 Oct. 1;
67(19):9371-9; Varghese S, et al., Clin Cancer Res. 2006 May 1;
12(9):2919-27; Qiao J, et al., Gene Ther. 2006 October;
13(20):1457-70; Heinkelein M, et al., Cancer Gene Ther. 2005
December; 12(12):947-53). Many studies have demonstrated that viral
delivery systems of siRNAs results in effective RNAi responses in
vitro and in vivo (Anesti A M, et al., Nucleic Acids Res. 2008
August; 36(14):e86; Gorbatyuk M, et al., Vision Res. 2007 April;
47(9):1202-8; Scherr M, et al., Nucleic Acids Res. 2007;
35(22):e149; Chen W, et al., J. Virol. 2006 April; 80(7):3559-66;
Raoul C, et al., Nat Med. 2005 April; 11(4):423-8; Bromberg-White J
L, et al., J. Virol. 2004 May; 78(9):4914-6; Scherr M, et al., Cell
Cycle. 2003 May-June; 2(3):251-7). The present invention provides
construct plasmid vectors (pVir) that produce virus particles (or
pseudovirions) upon transfection into mammalian cells. These
viruses carry biologically active RNAs targeting genes of interest
as part of a partial viral genome, allowing for expression of those
inhibitory sequences by either viral or host expression machinery.
When viral packaging cells are added to target cells or tissues,
the delivered RNAs can then modulate gene expression within each
infected target cell. For replication competent virus, a suicide
gene is added to the viral sequence such that viral replication can
be inhibited by the addition of a prodrug. This allows use of the
prodrug to prevent uncontrolled viral replication. For replication
defective virus, virus particles are produced exclusively in the
packaging cells for distribution to surrounding tissues; packaged
viral genomes include the biologically active RNAs but lack the
structural genes required for viral particle formation. This
arrangement prevents uncontrolled replication of the virus. This
system takes advantage of the highly efficient viral infection
efficiency and replication machinery to deliver and amplify the
inhibitory signal. As such, this approach is a direct compliment to
our plasmid based bioreactor delivery system.
[0260] In order for the viral packaging cell to function as a
delivery system, the viral particles must package and distribute a
biological signal, for example an inhibitory signal. This
biological signal could take the form of the biological RNA itself
or a DNA molecule encoding the biological RNA. Backbone vectors for
construction of viral based delivery systems therefore include both
DNA and RNA viruses, the former including appropriate promoters and
terminators for expression, the latter providing efficient Dicer
substrates. RNA viruses need only deliver the partial viral genome
(including the biological RNA) to the cytoplasm of the target cell;
DNA viruses require delivery of the DNA genome to the nucleus for
transcription of the biological RNA from the DNA template. Whereas
cytoplasmic delivery can be more efficient with the RNA viruses,
nuclear delivery provides opportunity for additional amplification
as multiple biologically active RNAs can be produced from a single
template molecule.
[0261] Viral packaging cells are generated by transfection of
recipient cells with plasmids encoding for the two independent
viral RNAs, one encoding the virus structural genes, the other
encoding the non-structural genes and the biologically active RNA
molecule. Successful co-transfection of both plasmids yields
packaging cells capable of producing replication defective viral
particles. Packaging of the DNA or RNA viral genome is driven by
the natural viral process, as is the secretion from the packaging
cell and import into the target cell. Once inside the target cell,
cellular mechanisms take over the specific biological process
depending on the identity of the particular biological molecule.
This delivery system is capable of accommodating any of the
biologically active RNAs described herein that act to modulate gene
expression of the target cell.
[0262] Viral based delivery can be combined with protein based
delivery in DNA viruses such that the initial transfection with
pVir plasmids results in production of viruses carrying both the
expression cassette for the biologically activeRNA and the
expression cassette for the fusion protein. In this aspect, the
viruses released from the viral packaging cells infect primary
target cells and transform them into protein based bioreactor
cells. These bioreactor cells then produce both the fusion protein
and the biologically active RNA for secretion and distribution to
secondary target cells. The expression cassettes for the
biologically active RNA and the fusion protein can be any of the
expression cassettes described herein.
[0263] Viral Backbones
[0264] Both DNA and RNA viruses are utilized as potential carriers
for inhibitory signals. A number of commonly used viral vectors are
appropriate for this type of application and have been
characterized in both in vitro and in vivo applications as
described above. Application of a particular viral system depends
on the desired target cells and can vary from tumor specific
delivery of the Sindbis virus particle through specific
interactions with the overexpressed laminin receptor (Tseng J C, et
al., Gene Ther. 2009 February; 16(2):291-6; Tseng J C, et al., J
Natl Cancer Inst. 2002; 94: 1790-1802) to non-specific delivery to
a broad spectrum of tissues as with the Foamy virus particle
(Heinkelein M, et al., Cancer Gene Ther. 2005 December;
12(12):947-53; Falcone V, et al., Curr Top Microbiol Immunol. 2003;
277: 161-180). Biological RNAs are intergrated into the expression
cassette for the non-structural viral genes for eventual packaging
into the replication defective viral particles.
[0265] In cases where gene knockdown is needed but lysis of the
target cell is undesirable, the use of replication defective
viruses is appropriate. These viruses efficiently deliver their
nucleic acid cargo to the interior of the cell, including the
biological RNA template or molecule. However, given that the
delivered nucleic acid does not contain a complete genome capable
of producing new virus particles, there is no viral replication or
subsequent cell lysis. In cases where lysis of the target cells is
desirable, such as cancer cells, the use of replication competent
oncolytic viruses may be most appropriate. These viruses are
selectively replicated in cancer target cells leading to their
eventual lysis (Ring C J, J Gen Virol. 2002 March; 83(Pt
3):491-502, Varghese S, et al., Cancer Res. 2007 Oct. 1;
67(19):9371-9; Varghese S, et al., Clin Cancer Res. 2006 May 1;
12(9):2919-27; Reinblatt M. et al., Surgery 2004; 136: 579-584).
The use of viruses that are capable of infecting human cells but do
not normally do so, such as viruses from other primates (Lund P E,
et al., Pharm Res. 2009 Dec. 9; Lund P E, et al., Pharm Res. 2009
Dec. 9; Heinkelein M, et al., Cancer Gene Ther. 2005 December;
12(12):947-53; Falcone V, et al., Curr Top Microbiol Immunol. 2003;
277: 161-180), can be useful in avoiding neutralizing antibodies
that can exist for viruses to which humans are natural hosts.
[0266] Application of Viral Packaging Cells In Vitro
[0267] Viral particles produced in viral packaging cells grown in
vitro are ultimately released from the packaging cells into the
culture media. These particles are routinely collected from growth
media, concentrated and used as transfection reagents for
biologically active RNAs (Heinkelein M, et al., Cancer Gene Ther.
2005 December; 12(12):947-53; Anesti A M, et al., Nucleic Acids
Res. 2008 August; 36(14):e86; Gorbatyuk M, et al., Vision Res. 2007
April; 47(9):1202-8; Scherr M, et al., Nucleic Acids Res. 2007;
35(22):e149; Chen W, et al., J Virol. 2006 April; 80(7):3559-66;
Raoul C, et al., Nat. Med. 2005 April; 11(4):423-8; Bromberg-White
J L, et al., J Virol. 2004 May; 78(9):4914-6; Scherr M, et al.,
Cell Cycle. 2003 May-June; 2(3):251-7). It may be possible to
infect target cells growing in culture without any processing of
the media from the viral packaging cells, by physically separating
the viral production and target cells yet allowing the two cultures
to share a common media. This is achieved using inserts designed to
fit in cell culture plates or by manual transfer of media from
production to target cells. In this case, the identity of the
packaging cells is optimized for virus production only. The viral
backbone is chosen to optimize particle stability in the cell
culture media and the highest possible titer without
concentration.
[0268] Viral packaging cells are also be used to transfect cells
growing in vitro by direct addition of the packaging cells to the
target cells. In one aspect, the viral delivered biological RNAs
(without intermediate concentration steps) are directly transferred
using the described type of co-culturing of viral production cells
and target cells transfected with reporter plasmids. The presence
of a specific reporter requires no distinction of viral production
and target cells and instead provides a direct readout of viral
based delivery of the biologically active RNAs. When using viral
systems to target endogenous genes, the readout for modulation of
gene expression by the biologically active RNA must be unique to
the target cell and not shared by the viral production cell,
similar to the experiments with the protein based bioreactor cells.
Recipient cells for the viral delivery system are dictated by the
identity of the target cells, so that species specific readout
simplifies analysis of the mRNA and protein knockdown. The optimal
ratio of viral packaging cells to target cells is determined
empirically for each combination of target cells and target
genes.
[0269] Modulation of Gene Expression In Vivo
[0270] Application of the viral packaging cells to in vivo systems
follow methods of transkaryotic implantation developed for the
overexpression of protein molecules in mouse model systems. As with
the protein based bioreactor cells, an in vivo test system
utilizing co-implantation of mouse tumor cell lines (SCCVII or
Renka) with viral packaging cells of mouse origin (see Examples 29
and 30) is used. A mixture of these cell types is implanted into
mice by subcutaneous injection into the rear flanks of the animal.
Viral particles deliver shRNAs targeting VEGF or Mmp2. Activity is
assayed by successful knockdown of the target gene in the region of
implantation or by physiological effects on tumor growth and
metastasis.
[0271] Viral packaging cells of mouse origin (NIH3T3 fibroblasts or
mESCs) is also implanted into mice to assay viral secretion and
delivery to surrounding mouse tissues. In this case, viral
particles containing biologically active RNA molecules target the
endogenous tissues of mouse models for human disease (see Examples
31-32). Relevant disease tissues are collected from each animal and
target gene expression is assessed at the transcript level using
RT-PCR or at the protein level using ELISA assays. Physiological
assays of disease progression is also measured and compared among
treated and non-treated control mice in order to assess both the
function of the viral based delivery system and the efficacy of the
gene target to treatment of the disease.
[0272] Kits
[0273] The invention further provides kits that can be used in the
methods described herein. For example, the invention provides kits
for constructing an expression vector, wherein the expression
vector expresses an RNA-protein complex of the invention. In one
embodiment, the kit comprises a first polynucleotide that encodes a
nucleic acid molecule comprising a recognition RNA sequence and
optionally a terminal minihelix sequence (hereinafter referred to
as the "RNA sequence") and a second polynucleotide that encodes a
polypeptide comprising an RNA binding domain and optionally one or
more transport peptide sequences (selected from a non-classical
secretory domain, a cell penetrating peptide, a receptor binding
domain, and an endosomal release domain (hereinafter referred to as
the "protein sequence"). In another embodiment, the kit
additionally comprises a third polynucleotide that encodes a
nucleic acid molecule comprising one or more biologically active
RNA sequences targeted to Dicer and/or Drosha (hereinafter referred
to as "Dicer/Drosha sequence").
[0274] Thus, in one embodiment, the kit further comprises one or
more primer sequences for amplifying the polynucleotide encoding
the RNA sequence (including the RNA binding sequence(s)). In one
embodiment, the primer sequence(s) comprises one or more sequences
complementary to the polynucleotide encoding the RNA sequence
(including the RNA binding sequence(s)), one or more restriction
enzyme site sequences, and optionally one or more sequences
comprising at least four GC base pairs. In another embodiment, the
kit additionally comprises a promoter sequence suitable for
expressing the polynucleotide encoding the RNA sequence (including
the RNA binding sequence(s)). In another embodiment, the kit
additionally comprises a termination sequence suitable for
expressing the polynucleotide encoding the RNA sequence (including
the RNA binding sequence(s)). In another embodiment, the kit
additionally comprises one or more primer sequences for amplifying
the polynucleotide encoding the protein sequence. In one
embodiment, the primer sequence(s) comprises one or more sequences
complementary to the polynucleotide encoding the protein sequence,
one or more restriction enzyme site sequences, and optionally one
or more sequences comprising at least four GC base pairs. In
another embodiment, the primer sequence(s) further comprises one or
more initiation codon sequences and one or more translational start
site sequences. In another embodiment, the kit additionally
comprises a promoter sequence suitable for expressing the
polynucleotide encoding the protein sequence. In another
embodiment, the kit additionally comprises a termination sequence
suitable for expressing the polynucleotide encoding the protein
sequence.
[0275] In alternate embodiments, the kit comprises a polynucleotide
comprising a recognition RNA sequence, optionally a terminal
minihelix sequence, optionally one or more biologically active RNA
sequences, one or more primer sequences, one or more promoter
sequences and one or more termination sequences. In one embodiment,
the polynucleotide comprises one or more biologically active RNA
sequences, wherein the biologically active RNA is selected from a
ribozyme, antisense nucleic acid, allozyme, aptamer, short
interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA
(miRNA), short hairpin RNA (shRNA), and a transcript encoding one
or more biologically active peptides. The biologically active RNA
can be targeted to any gene target of interest, including, for
example, VEGF, VEGFR, MMP2, Cav-1, EGFR, H-RAs, Bcl-2, Survivin,
FAK, STAT3, Her-3, Beta-catenin, hRET Receptor Tyrosine Kinase. In
another embodiment, polynucleotide does not include a biologically
active RNA sequence, which sequence is supplied by the individual
user of the kit. In one embodiment, the primer sequence(s)
comprises one or more sequences complementary to the polynucleotide
encoding the RNA sequence (including the biologically active RNA),
one or more restriction enzyme site sequences, and optionally one
or more sequences comprising at least four GC base pairs. In
another of the alternate embodiments, the kit further comprises a
polynucleotide comprising an RNA binding domain, one or more
sequences selected from a non-classical secretory domain, a cell
penetrating peptide, a receptor binding domain, an endosomal
release domain, one or more primer sequences, one or more promoter
sequences, and one or more termination sequences. In one
embodiment, the primer sequence(s) comprises one or more sequences
complementary to the polynucleotide encoding the protein sequence,
one or more restriction enzyme site sequences, optionally one or
more sequences comprising at least four GC base pairs, one or more
initiation codon sequences, and one or more translational start
site sequences. In another alternate embodiment, the kit also
comprises a polynucleotide comprising one or more biologically
active RNA sequences targeted to Dicer and/or Drosha, one or more
primer sequences, one or more promoter sequences and one or more
termination sequences.
[0276] In any of the described kit embodiments, the polynucleotide
encoding the RNA sequence (including the biologically active RNA)
can comprise a sequence wherein the recognition RNA sequence, the
individual biologically active RNA sequences, the optional terminal
minihelix sequence, and any other included sequences are joined
directly or are joined with the addition of one or more intervening
or additional sequences. In any of the described kit embodiments,
the polynucleotide encoding the protein sequence can comprise a
sequence wherein the RNA binding domain and the non-classical
secretory domain, cell penetrating peptide, receptor binding
domain, and endosomal release domain sequences and any other
included sequences are joined directly or are joined with the
addition of one or more intervening or additional sequences. Thus,
in certain embodiments, the kit additionally comprises linker
sequences for joining the various sequences and domains of the
polynucleotide encoding the RNA sequence and the polynucleotide
encoding the protein sequence.
[0277] In any of the described kit embodiments, the recognition RNA
sequence can be selected from a U1 loop, Group II intron, NRE stem
loop, S1A stem loop, bacteriophage BoxBR, HIV Rev response element,
AMVCP recognition sequence, and ARE sequence. In any of the
described kit embodiments, the RNA binding domain can be selected
from a U1A, CRS1, CRM1, Nucleolin RBD12, hRBMY, Bacteriophage
Protein N, HIV Rev, AMVCP, and tristetrapolin sequence. In any of
the described kit embodiments, the cell penetrating peptide can be
selected from a penetratin, transportan, MAP, HIV TAT, Antp, Rev,
FHV coat protein, TP10 and pVEC sequence. In any of the described
kit embodiments, the non-classical secretory domain can be selected
from Galcetin-1 peptide, Galectin-3 peptide, IL-1.alpha.,
IL-1.beta., HASPB, HMGB1, FGF-1, FGF-2, IL-2 signal, secretory
transglutaminase, annexin-1, HIV TAT, Herpes VP22, thioredoxin,
Rhodanese, and plasminogen activator signal sequences. In any of
the kit embodiments, the promoter is a Pol II promoter.
Non-limiting examples of suitable Pol II promoters include, but are
not limited to, Simian Virus 40 (SV40), Cytomegalovirs (CMV),
.beta.-actin, human albumin, human HIF-.alpha., human gelsolin,
human CA-125, ubiquitin, and PSA promoters. In another embodiment,
the promoter is a Pol III promoter. Examples of suitable Pol III
promoters include, but are not limited to, human H1 and human U6
promoters. Non-limiting examples of suitable termination sequences
include, but are not limited to, the human growth hormone (hGH)
polyadenylation sequence, the bovine growth hormone (BGH)
polyadenylation sequence, the Simian Virus 40 (SV40) large T
polyadenylation sequence, and the Herpes Simplex Virus Thymidine
Kinase (HSV-tk) polyadenylation sequence.
[0278] In yet another embodiment, the kit further comprises one or
more backbone vectors into which the polynucleotide encoding the
RNA sequence (including the biologically active RNA) and/or the
polynucleotide encoding the protein sequence and/or the
polynucleotide encoding the Dicer/Drosha sequence can be inserted.
In one embodiment, the polynucleotide encoding the RNA sequence is
inserted into a first backbone vector and the polynucleotide
encoding the protein sequence is inserted into a second backbone
vector. In another embodiment, the polynucleotide encoding the RNA
sequence and the polynucleotide encoding the protein sequence is
inserted into a single backbone vector. In one embodiment, the
polynucleotide encoding the Dicer/Drosha sequence can be inserted
into a third backbone vector. In another embodiment, the
polynucleotide encoding the Dicer/Drosha sequence can be inserted
into the same vector as the polynucleotide encoding the RNA
sequence. Non-limiting examples of suitable backbone vectors
include pCI, pET, pSI, pcDNA, pCMV, etc. In any of the above
embodiments, the backbone vector additionally comprises a pUC
origin of replication. In one embodiment, the backbone vector
additionally comprises one or more drug resistance genes selected
from a kanamycin, ampicillin, puromycin, tetracycline, and
chloramphenicol resistant genes, as well as any other drug
resistant genes known and described in the art.
[0279] In other embodiments, the kit additionally comprises
buffers, enzymes, and solutions useful for amplifying, cloning
and/or expressing the polynucleotide encoding the RNA (including
the biologically active RNA) sequence, the polynucleotide encoding
the protein sequence, and the polynucleotide encoding the
Dicer/Drosha sequence, including, for example, one or more
restriction enzymes, phosphatases, kinases, ligases, and
polymerases.
[0280] In another embodiment, the kit additionally comprises
instructions for constructing the expression vectors, including,
for example, polynucleotide sequence maps and plasmid maps.
[0281] In another embodiment, the kit additionally comprises
materials for packaging the kits for commercial use.
[0282] In addition, the invention provides kits comprising
expression vectors useful for modulating the expression of a target
gene. The kit provides one or more expression vectors that produce
an RNA-protein complex of the invention that can be used to
modulate gene expression in vivo, ex vivo, and in vitro. In one
embodiment, the kit comprises separate expression vectors for
expressing the RNA portion of the RNA-protein complex and the
fusion protein portion of the RNA-protein complex. One of the
advantages of the kits comprising separate expression vectors for
the RNA portion and the protein portion of the RNA-protein complex
is that the activity of the biologically active RNA can be verified
by transfecting the vector comprising the biologically active RNA
into target cells. In the absence of the vector expressing the
fusion protein, the gene-modulation of the vector expressing the
biologically active RNA can be verified directly in the target
cell. In another embodiment, the kit comprises a single expression
vector for expressing the RNA-protein complex.
[0283] In one embodiment, the kit provides an expression vector
comprising one or more biologically active RNA sequences directed
to a target gene, a recognition RNA sequence, optionally a terminal
minihelix sequence, one or more promoter sequences, one or more
termination sequences, restriction enzyme sites, primer sequences,
and optionally GC base pair sequences, wherein the biologically
active RNA sequence(s), the recognition RNA sequence, and the
optional terminal minihelix sequence are downstream of a promoter
sequence. The biologically active RNA can be any biologically
active RNA described herein or otherwise known in the art. The
biologically active RNA sequence can be selected from a ribozyme,
antisense nucleic acid, allozyme, aptamer, short interfering RNA
(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short
hairpin RNA (shRNA), and a transcript encoding one or more
biologically active peptides. The biologically active RNA can be
targeted to any gene target of interest, including, for example,
VEGF, VEGFR, MMP2, Cav-1, EGFR, H-RAs, Bcl-2, Survivin, FAK, STAT3,
Her-3, Beta-catenin, hRET Receptor Tyrosine Kinase. In another
embodiment, the expression vector does not include a biologically
active RNA sequence, which sequence is supplied by the individual
user of the kit. Thus, in one embodiment, the kit provides an
expression vector comprising a recognition RNA sequence, optionally
a terminal minihelix sequence, one or more promoter sequences, one
or more termination sequences, restriction enzyme sites, primer
sequences, and optionally GC base pair sequences, wherein the
recognition RNA sequence and the optional terminal minihelix
sequence are downstream of a promoter sequence. The restriction
enzymes sites are located so as to provide convenient cloning sites
for insertion of the user's biologically active RNA sequence. In
another alternate embodiment, the kit also comprises a
polynucleotide comprising one or more biologically active RNA
sequences targeted to Dicer and/or Drosha, one or more primer
sequences, one or more promoter sequences and one or more
termination sequences.
[0284] In any of the above embodiments, the recognition RNA
sequence can be selected from a U1 loop, Group II intron, NRE stem
loop, S1A stem loop, Bacetriophage BoxB, HIV Rev response element,
AMVCP recognition sequence, and ARE sequence. In one embodiment,
the promoter sequence is a polIII promoter. Non-limiting examples
of suitable polIII promoters include human U6 polIII promoter and
human H1 polIII promoter. In one embodiment, the promoter sequence
is a polII promoter. Non-limiting examples of suitable polII
promoters include SV40, .beta.-actin, human albumin, human
HIF-.alpha., human gelsolin, human CA-125, human ubiquitin, PSA,
and cytomegalovirus (CMV) promoters. In one embodiment, the
biologically active RNA sequence and the recognition RNA sequence
are operably linked to the promoter sequence. In one embodiment,
the termination sequence is a Pol-III polyT termination
sequence.
[0285] In any of the above embodiments, the expression vector
additionally comprises a pUC origin of replication. In any of the
above embodiments, the expression vector additionally comprises one
or more drug resistance genes. Examples of suitable drug resistant
genes include, but are not limited to, kanamycin, ampicillin,
puromycin, tetracycline, and chloramphenicol resistant genes, as
well as any other drug resistant genes known and described in the
art.
[0286] In one embodiment, the kit additionally comprises an
expression vector comprising an RNA binding domain, and one or more
sequences selected from a cell penetrating peptide, a non-classical
secretory domain, a receptor binding domain, an endosomal release
domain, and a fusogenic peptide, and additionally comprises one or
more promoter sequences, one or more termination sequences,
restriction enzyme sites, primer sequences, optionally GC base pair
sequences, an initiation codon, and a translational start site,
wherein the RNA binding domain and the cell penetrating peptide,
non-classical secretory domain, receptor binding domain, endosomal
release domain, and fusogenic peptide are downstream of the
promoter sequence. In one embodiment, the promoter sequence is a
Pol II promoter. Non-limiting examples of suitable polII promoters
include SV40, .beta.-actin, human albumin, human HIF-.alpha., human
gelsolin, human CA-125, human ubiquitin, PSA, and cytomegalovirus
(CMV) promoters. The termination sequence can be a polyadenylation
sequence, for example, a poly adenylation sequence derived from
hGH. In certain embodiments, the RNA binding domain comprises an
amino acid sequence selected from a U1A, CRS1, CRM1, Nucleolin
RBD12, hRBMY, Bacteriophage Protein N, HIV Rev, AMVCP, and
tristetrapolin amino acid sequence. In certain embodiments, the
cell penetrating peptide comprises an amino acid sequence selected
from a penetratin, transportan, MAP, HIV TAT, Antp, Rev, FHV coat
protein, TP10, and pVEC amino acid sequence. In certain
embodiments, the non-classical secretory domain comprises an amino
acid sequence selected from Galcetin-1 peptide, Galectin-3 peptide,
IL-1.alpha., IL-1.beta., HASPB, HMGB1, FGF-1, FGF-2, IL-2 signal,
secretory transglutaminase, annexin-1, HIV TAT, Herpes VP22,
thioredoxin, Rhodanese, and plasminogen activator signal amino acid
sequences.
[0287] In any of the above embodiments, the expression vector
additionally comprises a pUC origin of replication. In one
embodiment, the expression vector additionally comprises one or
more drug resistance genes selected from a kanamycin, ampicillin,
puromycin, tetracycline, and chloramphenicol resistant genes, as
well as any other drug resistant genes known and described in the
art.
[0288] In one embodiment, the kit can optionally further comprise
an expression vector comprising one or more biologically active RNA
sequences, optionally a terminal minihelix sequence, one or more
promoter sequences, one or more termination sequences, restriction
enzyme sites, primer sequences, and optionally GC base pair
sequences, wherein the biologically active RNA sequence(s) and the
optional terminal minihelix sequence are downstream of a promoter
sequence and wherein the biologically active RNA sequence(s) are
targeted to Dicer and/or Drosha. In certain embodiments, the
biologically active RNA sequence is selected from a ribozyme,
antisense nucleic acid, allozyme, aptamer, short interfering RNA
(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short
hairpin RNA (shRNA), and a transcript encoding one or more
biologically active peptides. In one embodiment, the promoter
sequence(s) is a polIII promoter, including for example, a human U6
polIII promoter and human H1 polIII promoter. In one embodiment,
the promoter sequence is a polII promoter, including, for example,
SV40, .beta.-actin, human albumin, human HIF-.alpha., human
gelsolin, human CA-125, human ubiquitin, PSA, and cytomegalovirus
(CMV) promoters. In one embodiment, the termination sequence(s) is
a Pol-III polyT termination sequence. In any of the above
embodiments, the expression vector additionally comprises a pUC
origin of replication. In one embodiment, the expression vector
additionally comprises one or more drug resistance genes selected
from a kanamycin, ampicillin, puromycin, tetracycline, and
chloramphenicol resistant genes, as well as any other drug
resistant genes known and described in the art.
[0289] In another embodiment, the kit additionally comprises
instructions and materials for packaging the kits for commercial
use.
[0290] Alternatively, the kit comprises a single expression vector
encoding an RNA-protein complex of the invention. In one
embodiment, the kit comprises an expression vector comprising a
first expression cassette, a second expression cassette, and
optionally a third expression cassette. The first expression
cassette comprises one or more biologically active RNA sequences
directed to a target gene(s), a recognition RNA sequence,
optionally a terminal minihelix sequence, one or more promoter
sequences, one or more termination sequences, restriction enzyme
sites, primer sequences, and optionally GC base pair sequences,
wherein the biologically active RNA sequence(s), the recognition
RNA sequence, and the optional terminal minihelix sequence are
downstream of a promoter sequence. In certain embodiments, the
biologically active RNA sequence is selected from a ribozyme,
antisense nucleic acid, allozyme, aptamer, short interfering RNA
(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short
hairpin RNA (shRNA), and a transcript encoding one or more
biologically active peptides. The target gene can be any target
gene, including, for example, Mmp2, Vascular Endothelial Growth
Factor (VEGF), Vascular Endothelial Growth Factor Receptor (VEGFR),
Cav-1, Epidermal Growth Factor Receptor (EGFR), H-Ras,Bcl-2,
Survivin, FAK, STAT-3, HER-3, Beta-Catenin, and Src gene targets.
In certain embodiments, the recognition RNA sequence is selected
from a U1 loop, Group II intron, NRE stem loop, S1A stem loop,
Bacetriophage BoxBR, HIV Rev response element, AMVCP recognition
sequence, and ARE sequence. In one embodiment, the promoter
sequence is a polIII promoter, including, for example, a promoter
selected from a human U6 polIII promoter and human H1 polIII
promoter. In one embodiment, the promoter sequence is a porn
promoter, including, for example, a promoter selected from an SV40,
.beta.-actin, human albumin, human HIF-.alpha., human gelsolin,
human CA-125, human ubiquitin, PSA, and cytomegalovirus (CMV)
promoters. In one embodiment, the termination sequence is a Pol-III
polyT termination sequence.
[0291] The expression vector of the kit further comprises a second
expression cassette, wherein the second expression cassette
comprises an RNA binding domain sequence, one or more sequences
selected from a cell penetrating peptide, a non-classical secretory
domain, a receptor binding domain, an endosomal release domain, and
a fusogenic peptide, one or more promoter sequences, one or more
termination sequences, restriction enzyme sites, primer sequences,
GC base pair sequences, an initiation codon, and translational
start site, wherein the RNA binding domain and the cell penetrating
peptide, non-classical secretory domain, receptor binding domain,
endosomal release domain, and fusogenic peptide are downstream of a
promoter sequence. In certain embodiments, the RNA binding domain
is selected from a U1A, CRS1, CRM1, Nucleolin RBD12, hRBMY,
Bacteriophage Protein N, HIV Rev, AMVCP, and tristetrapolin
sequence. In certain embodiments, the cell penetrating peptide is
selected from a penetratin, transportan, MAP, HIV TAT, Antp, Rev,
FHV coat protein, TP10, and pVEC amino acid sequence. In certain
embodiments, the non-classical secretory domain is selected from a
Galectin-1 peptide, Galectin-3 peptide, IL-1.alpha., IL-1.beta.,
HASPB, HMGB1, FGF-1, FGF-2, IL-2 signal, secretory
transglutaminase, annexin-1, HIV TAT, Herpes VP22, thioredoxin,
Rhodanese, and plasminogen activator signal sequence. In one
embodiment, the promoter sequence is a Pol II promoter, including,
for example, a promoter selected from an SV40, .beta.-actin, human
albumin, human HIF-.alpha., human gelsolin, human CA-125, human
ubiquitin, PSA, and cytomegalovirus (CMV) promoters. In one
embodiment, the termination sequence is a polyadenylation sequence.
In one embodiment, the poly adenylation sequence is derived from
hGH.
[0292] The expression vector of the kit optionally further
comprises a third expression cassette, wherein the third expression
cassette comprises one or more biologically active RNA sequences
and optionally a terminal minihelix sequence, one or more promoter
sequences, one or more termination sequences, restriction enzyme
sites, primer sequences, and optionally GC base pair sequences,
wherein the biologically active RNA sequence(s) and the optional
terminal minihelix sequence are downstream of the promoter
sequence. In certain embodiments of the above-described expression
vectors, the biologically active RNA sequence is selected from a
ribozyme, antisense nucleic acid, allozyme, aptamer, short
interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA
(miRNA), short hairpin RNA (shRNA), and a transcript encoding one
or more biologically active peptides. In one embodiment, one or
more of the biologically active RNA sequences is directed to Dicer
and/or Drosha. In one embodiment, the promoter sequence is a polIII
promoter. Non-limiting examples of suitable polIII promoters
include human U6 polIII promoter and human H1 polIII promoter. In
one embodiment, the promoter sequence is a polII promoter.
Non-limiting examples of suitable poll promoters include SV40,
.beta.-actin, human albumin, human HIF-.alpha., human gelsolin,
human CA-125, human ubiquitin, PSA, and cytomegalovirus (CMV)
promoters. In one embodiment, the biologically active RNA sequence
is operably linked to the promoter sequence. In one embodiment, the
termination sequence is a Pol-III polyT termination sequence.
[0293] The expression vector additionally comprises a pUC origin of
replication. In one embodiment, the expression vector additionally
comprises one or more drug resistance genes selected from a
kanamycin, ampicillin, puromycin, tetracycline, and chloramphenicol
resistant gene, as well as any other drug resistant genes known and
described in the art.
[0294] In an alternate embodiment, the kit comprises an expression
vector comprising a first expression cassette, a second expression
cassette, and optionally a third expression cassette, wherein the
first expression cassette comprises a recognition RNA sequence,
optionally a terminal minihelix sequence, one or more promoter
sequences, one or more termination sequences, restriction enzyme
sites, primer sequences, and optionally GC base pair sequences, and
wherein the recognition RNA sequence and the optional terminal
minihelix sequence are downstream of a promoter sequence. The kit
does not include a biologically active RNA sequence, which sequence
is supplied by the individual user of the kit. The kit optionally
comprises one or more primer sequences comprising restriction
enzymes sites which can be ligated to the biologically active RNA
sequence for convenient cloning into the expression vector. The
second expression cassette and optional third expression cassette
can be any of the second and third expression cassettes described
above.
[0295] In an alternate embodiment, the kit comprises an expression
vector comprising the second expression cassette and optionally the
third expression cassette. The kit additionally comprises an
isolated polynucleotide comprising a first expression cassette that
can be ligated into the expression vector, wherein the first
expression cassette comprises a recognition RNA sequence,
optionally a terminal minihelix sequence, one or more promoter
sequences, one or more termination sequences, restriction enzyme
sites, primer sequences, and optionally GC base pair sequences, and
wherein the recognition RNA sequence, and the optional terminal
minihelix sequence are downstream of a promoter sequence. The kit
does not include a biologically active RNA sequence, which sequence
is supplied by the individual user of the kit. The kit optionally
comprises one or more primer sequences which can be ligated to the
biologically active RNA sequence for convenient insertion into the
first expression cassette. The first expression cassette can then
be cloned into the expression vector comprising the second
expression cassette and the third expression cassette. The kit
optionally comprises one or more primer sequences comprising
restriction sites compatible with the expression vector which can
be ligated to the first expression cassette for convenient cloning
into the expression vector. The second expression cassette and
third expression cassette can be any of the second and third
expression cassettes described above. In embodiments wherein the
expression vector comprises only the second expression cassette,
the kit can additionally comprise an isolated polynucleotide
comprising a third expression cassette that can be ligated into the
expression vector. The third expression cassette can be any of the
third expression cassettes described above. The kit optionally
comprises one or more primer sequences comprising restriction sites
compatible with the expression vector which can be ligated to the
third expression cassette for convenient cloning into the
expression vector.
[0296] In any of these embodiments, the expression vector
additionally comprises a pUC origin of replication. In one
embodiment, the expression vector additionally comprises one or
more drug resistance genes selected from a kanamycin, ampicillin,
puromycin, tetracycline, and chloramphenicol resistant gene, as
well as any other drug resistant genes known and described in the
art.
[0297] The invention also provides a kit comprising one or more
bioreactor cells that produce an RNA-protein complex of the
invention that can be used to modulate gene expression in vivo, ex
vivo, and in vitro. The invention provides a solution of bioreactor
cells that produce and secrete an RNA-protein complex comprising
one or more biologically active RNA sequences, a recognition RNA
sequence, optionally a terminal minihelix sequence, an RNA binding
domain sequence, and one or more sequences selected from a
cell-penetrating peptide, non-classical secretory domain, endosomal
release domain, receptor binding domain, and fusogenic peptide
sequence. In one embodiment, the bioreactor cell produces an
RNA-protein complex comprising one or more biologically active RNA
sequences, a recognition RNA sequence, an optional terminal
minihelix sequence, an RNA binding domain sequence, and a
cell-penetrating peptide sequence. In another embodiment, the
bioreactor cell produces an RNA-protein complex comprising one or
more biologically active RNA sequences, a recognition RNA sequence,
an optional terminal minihelix sequence, an RNA binding domain
sequence, and a non-classical secretory domain sequence. In yet
another embodiment, the bioreactor cell produces an RNA-protein
complex comprising one or more biologically active RNA sequences, a
recognition RNA sequence, an optional terminal minihelix sequence,
an RNA binding domain sequence, a cell-penetrating peptide
sequence, and a non-classical secretory domain sequence.
[0298] In certain embodiments of the above-described kits
comprising bioreactor cells, the biologically active RNA
sequence(s) is selected from a ribozyme, antisense nucleic acid,
allozyme, aptamer, short interfering RNA (siRNA), double-stranded
RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), and a
transcript encoding one or more biologically active peptides. The
biologically active RNA sequence(s) can be targeted to any gene,
including but are not limited to, Mmp2, Vascular Endothelial Growth
Factor (VEGF), Vascular Endothelial Growth Factor Receptor (VEGFR),
Cav-1, Epidermal Growth Factor Receptor (EGFR), H-Ras,Bcl-2,
Survivin, FAK, STAT-3, HER-3, Beta-Catenin, and Src gene targets.
In certain embodiments of the above-described cells, the
recognition RNA sequence is selected from a U1 loop, Group II
intron, NRE stem loop, S1A stem loop, Bacteriophage BoxBR, HIV Rev
response element, AMVCP recognition sequence, and ARE sequence. In
certain embodiments of the above-described cells, the RNA binding
domain is selected from a U1A, CRS1, CRM1, Nucleolin RBD12, hRBMY,
Bacteriopage Protein N, HIV Rev, AMVCP, and tristetrapolin
sequence. In certain embodiments of the above-described cells, the
cell penetrating peptide comprises a sequence selected from a
penetratin, transportan, MAP, HIV TAT, Antp, Rev, FHV coat protein,
TP10, and pVEC amino acid sequence. In certain embodiments of the
above-described cells, the non-classical secretory domain comprises
a sequence selected from a Galcetin-1 peptide, Galectin-3 peptide,
IL-1.alpha., IL-1.beta., HASPB, HMGB1, FGF-1, FGF-2, IL-2 signal,
secretory transglutaminase, annexin-1, HIV TAT, Herpes VP22,
thioredoxin, Rhodanese, and plasminogen activator signal
sequence.
[0299] Non-limiting examples of suitable cells include NIH 3T3,
Cos-1, Cos-7, SCCVII, HEK293, PC-12, Renka, A549, CT26, CHO, HepG2,
Jurkat, and HeLa cells, as well as any other cells known and
described in the art.
[0300] It will be clear that the invention may be practiced
otherwise than as particularly described in the foregoing
description and the following examples. Numerous modifications and
variations of the invention are possible in light of the teachings
herein and, therefore, are within the scope of the appended
claims.
EXAMPLES
Example 1
General Construction of a Bioreactor Plasmid of the Invention
[0301] Expression vectors are constructed from isolated plasmid
backbones and PCR amplified expression cassettes for both the RNA
(sec-RNA) and protein (fusion protein) components. Examples of
suitable backbone vectors include those derived from pCI, pET, pSI,
pcDNA, pCMV, etc. The expression vector should include at least the
following components: an origin of replication for preparation in
bacteria, an antibiotic selectable marker, a promoter for RNA
expression (Pol-II or Pol-III), a terminator sequence appropriate
to the promoter sequence, a promoter for fusion protein expression
and a poly-A tail sequence. One example of a suitable backbone
vector is selected from the various pEGEN backbone vectors
described herein, which are derived from pSI (Promega, product
#E1721), pCI (Promega, product #E1731), pVAX (Invitrogen, product
#12727-010) and other in house constructs. The pEGEN vectors, e.g.
pEGEN 1.1, pEGEN 2.1, pEGEN 3.1, and pEGEN 4.1, contain a pUC
origin of replication and a kanamycin resistance gene allowing the
vector to be replicated in bacteria and cultured in the presence of
kanamycin. Other suitable backbone vectors are well-known and
commercially available, for example, pCI, pSI, pcDNA, pCMV, etc.
The pEGEN vector is transformed into XL1-Blue competent cells via
standard heat shock methods. Transformed cells are selected by
growth on LB-Kanamycin plates, individual colonies are used to seed
5 mL LB-Kanamycin liquid cultures and grown overnight at 37.degree.
C. Resulting cultures are used to prepare purified plasmid stocks
using standard methods.
[0302] Expression cassettes for the protein components of the
bioreactor plasmid are prepared by PCR amplification of the
relevant sequences from cDNA clones using the appropriate forward
and reverse primers. Primers typically include sequences
complementary to the domain(s) of interest (e.g., RNA binding
domain, cell penetrating peptide, non-classical secretory domain,
endosomal release domain, receptor binding domain, fusogenic
peptide, etc.), sites for restriction enzymes used in the
subcloning, and at least four GC base pairs at the 5' end of each
primer to facilitate digestion with restriction enzymes. Other
useful primers can include sequences complementary to the domain(s)
of interest (e.g., RNA binding domain, cell penetrating peptide,
non-classical secretory domain, endosomal release domain, receptor
binding domain, fusogenic peptide, etc.), sites for restriction
enzymes used in the subcloning, and 15 bases of vector sequence
flanking the restriction site for use in recombination cloning
(In-fusion Advantage PCR cloning kit, Clontech, Catalog #639620).
Other suitable primers include sequences complementary to the
protein domain(s), sites for restriction enzymes used in subcloning
and six GC base pairs at the 5' end of each primer. Initiation
codons and optimized Kozak translational start sites are added to
each primer corresponding to the 5' end of the transcript to
promote translation of the N-terminal domains of each fusion
protein. Restriction sites are added to the primer corresponding to
the 3' end of the transcript to facilitate assembly of delivery
domains with RNA binding domains. A typical PCR reaction contains
10 mM Tris-HCl pH 9.0, 50 mM KCl, 1.5 mM MgCl.sub.2, 0.1% Triton
X-100, 200 .mu.M each dNTP, 1.0 .mu.M sense primer, 1.0 .mu.M
antisense primer, 100 ng DNA template and 1.0 U of Taq polymerase
per 50 .mu.L reaction. Reactions are cycled through 3 temperature
steps: a denaturing step at 95.degree. C. for 30 seconds, an
annealing step at 50.degree. C. to 60.degree. C. for 30 seconds and
an elongation step at 72.degree. C. for 1 minute. Typically, the
total number of cycles ranges from 20 to 35 cycles depending on the
specific amplification reaction.
[0303] Domains can be linked to one another directly or via
sequences encoding alpha helical linker or other linker domains.
These linkers provide separation between the two functional domains
to avoid possible steric issues. In each case, restriction
digestions of DNAs encoding each domain produce compatible ends for
directional ligation. A typical restriction digestion contains 10
mM Tris (pH 8.0), 100 mM NaCl, 5 mM MgCl.sub.2, 1 mM DTT, 0.1-1
unit of each restriction enzyme and 1 .mu.g of DNA and is digested
at 37.degree. C. for 1 hour. Products are purified on 2% agarose
gels run in 1.times.TAE and excised bands are recovered using
Qiagen's Qiaex II gel purification system. These expression
cassettes are cloned into the multiple cloning site of the pEGEN
vector using restriction enzymes matching the insert of interest. A
typical ligation reaction contains 30 mM Tris (pH 7.8), 10 mM
MgCl.sub.2, 10 mM DTT, 1 mM ATP, 100 ng DNA vector, 100 to 500 ng
DNA insert, 1 unit T4 DNA ligase and is ligated overnight at
16.degree. C. Another typical recombination reaction contains
1.times. In-fusion reaction buffer, 100 ng of linearized plasmid,
50-200 ng of insert, 1 unit of In-fusion enzyme, which is incubated
first at 37.degree. C. for 15 minutes and then at 50.degree. C. for
15 minutes. This process places the expression cassette downstream
of a strong Pol II promoter sequence and upstream of an hGH polyA
signal sequence. As shown in FIGS. 5-7, the Pol II promoter for
pEGEN 1.1 comprises an SV40 promoter, the Pol II promoter for pEGEN
2.1 comprises a chicken .beta.-actin promoter, and the Pol II
promoter for pEGEN 3.1 comprises a CMV promoter. Successful cloning
of the PCR product into the plasmid vector can be confirmed with
restriction mapping using enzymes with sites flanking the insertion
point and with PCR using primers specific to the insert sequence
(for example, see FIG. 15).
[0304] The vector comprising the fusion protein cassette can be can
be used to transfect cells in combination with a vector comprising
a Sec-RNA of the invention, described below.
[0305] Expression cassettes for the RNA components (e.g.,
recognition RNA sequence and biologically active RNA sequence,
including, for example, ribozymes, antisense nucleic acids,
allozymes, aptamers, short interfering RNA (siRNA), double-stranded
RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), and RNA
transcript encoding a biologically active peptide) of the
bioreactor plasmid are prepared by PCR amplification of the
relevant sequences from RNA expressing plasmids using the
appropriate forward and reverse primers. Primers include sequences
complementary to the biologically active RNA sequence(s), sites for
restriction enzymes used in subcloning and at least four GC base
pairs at the 5' end of each primer to facilitate digestion with
restriction enzymes. Other suitable primers can include sequences
complementary to the domain(s) of interest (e.g., RNA binding
domain, cell penetrating peptide, non-classical secretory domain,
endosomal release domain, receptor binding domain, fusogenic
peptide, etc.), sites for restriction enzymes used in the
subcloning, and 15 bases of vector sequence flanking the
restriction site for use in recombination cloning (In-fusion
Advantage PCR cloning kit, Clontech, Catalog #639620). In one
specific example, the primers include sequences complementary to
the biologically active RNA sequence(s), sites for restriction
enzymes used in subcloning and six GC base pairs at the 5' end of
each primer. The recognition RNA sequence is added to the primer
corresponding to the 5' end of the biologically active RNA sequence
in order to generate the Sec-RNA expression construct. This
expression construct is digested with appropriate restriction
enzymes for subcloning into the pEGEN4.1 construct, which places
the Sec-RNA expression cassette downstream from a strong Pol-III
promoter sequence (the human U6 promoter for pEGEN4.1, and the
human H1 promoter for pEGEN5.1) and upstream of a Pol III poly-T
termination sequence. See FIG. 8. Alternatively, the expression
construct is subcloned into the pEGEN5.1 construct, which places
the Sec-RNA expression cassette downstream from the human H1
promoter sequence (Pol-III promoter) and upstream of a Pol III
poly-T termination sequence. Alternatively, the Sec-RNA expression
cassette can be subcloned into pEGEN1.1, 2.1, or 3.1, which places
RNA expression under the control of the SV40, .beta.-actin, and CMV
Pol-II promoter, respectively, and terminates with a human GH
polyadenylation signal. Alternatively, the Sec-RNA expression
cassette can be subcloned into any of pEGEN 6.1-11.1.
[0306] The vector comprising the Sec-RNA expression cassette can be
used to transfect cells in combination with a vector comprising a
fusion protein of the invention, described above.
[0307] Successful cloning of the PCR product into the plasmid
vector can be confirmed with restriction mapping using enzymes with
sites flanking the insertion point and with PCR using primers
specific to the insert sequence. For example, FIG. 15 shows
restriction enzyme analysis (15 C and D) and PCR amplification
analysis (15E) of a sec-RNA plasmid (15C) and fusion protein
plasmids (15D and E). FIG. 15C shows the restriction enzyme
analysis of the pE3.1 Sec-Reporter, in which a novel EcoNI
restriction site is introduced with the RNA expressing insert.
FIGS. 15D and 15E show the restriction enzyme and PCR analyses,
respectfully, of two pE1 TAT-RBD plasmids. In FIG. 15C,
Sec-Reporter (-) refers to the pE3.1 Sec-Reporter plasmid only and
Sec-Reporter (+) refers to the pE3.1 Sec-Reporter plasmid with the
sec-RNA expressing insert. In FIGS. 15D and 15E, p1.1 refers to the
pE1.1 plasmid only, TAT(-) refers to the pE1.1 plasmid with the
fusion protein insert comprising a TAT cell penetrating peptide
fused to a Rev RNA binding domain, and TAT(+) refers to the pE1.1
plasmid with the fusion protein insert comprising a TAT cell
penetrating peptide fused to a Protein N RNA binding domain.
Restriction digestion of each plasmid with XcmI and AleI enzymes
(which flank the site of insertion) allows agarose gel analyses
which distinguishes between empty parent plasmid (a 99 bp product)
and successful subcloning of the insert (245 bp product). PCR
amplification of the insertion site using one primer annealing to
the coding strand of the fusion protein insert and a second primer
annealing to the non-coding strand of the polyA sequence produces a
416 bp product for a properly oriented insert. Plasmid insert
identity was confirmed through DNA sequencing.
[0308] In those embodiments of the invention wherein the Sec-RNA
expressing cassette and the fusion protein cassette are in a single
vector, the final subcloning step joins the fusion protein
expressing cassette with the Sec-RNA expressing cassette into a
single plasmid vector, the pBioR plasmid. In one embodiment, the
Sec-RNA expression cassette (e.g., primers, promoter, recognition
RNA sequence, biologically active RNA, and termination sequence) is
ligated into the pEGEN plasmid comprising the fusion protein to
generate the complete pBioR plasmid. Restriction sites flanking the
expression cassette, as shown in for example, the Sec-RNA in
pEGEN4.1 (FIG. 8) or pEGEN5.1 (not depicted) release the insert
from the plasmid, which is then purified on 2% agarose gels run in
1.times.TAB and excised bands are recovered using, for example,
Qiagen's Qiaex II gel purification system. The plasmid containing
the expression cassette for the fusion protein is digested with the
same restriction enzyme flanking the Sec-shRNA expression cassette.
The Sec-RNA expression cassette is then ligated into the plasmid
containing the fusion protein to generate the complete pBioR
plasmid.
Example 2
Construction of a Bioreactor Plasmid pBioR(1) with a Sec-shRNA
Delivered by a CPP-RBD Fusion Protein
[0309] An expression vector capable of expressing a bioreactor
fusion protein and a secreted shRNA (Sec-shRNA) is described here.
Production and delivery of Sec-shRNAs targeting any of the gene
targets listed in Table I and Table VII, as well as any other
target mRNAs, is accomplished with the plasmid pBioR(1), which is
constructed from two parent plasmids. The first parent plasmid,
pEGENFP, expresses the fusion protein and is constructed by cloning
a fusion protein cassette comprising an RNA binding domain sequence
from Table III and a cell penetrating peptide sequence from Table
IV into the multiple cloning site of a pEGEN vector from Table VIII
using the plasmids and methods described in Example 1. In one
embodiment, this process places the fusion protein cassette
downstream of a strong Pol II promoter sequence (chicken
.beta.-actin promoter) and upstream of an hGH polyA signal
sequence. The RNA binding domain and the cell penetrating peptide
fusion protein can be assembled with or without alpha helical
linker domains. This vector can be transfected into cells in
combination with a pEGENSR vector.
[0310] The second parent plasmid, pEGENSR, expresses the secreted
RNA and is constructed by cloning a secreted RNA cassette
comprising an RNA recognition element from Table II and a
biologically active RNA from Table I into the multiple cloning site
of the pEGEN4.1 or pEGEN5.1 vector (see Table VIII) using the
plasmids and methods described in Example 1. This process places
the Sec-RNA cassette downstream from a Pol III promoter (a human U6
promoter for pEGEN4.1, a human H1 promoter for pEGEN5.1) and
upstream of a Pol III poly-T termination sequence. This vector can
be transfected into cells in combination with a pEGENFP vector.
Alternatively, the expression cassette for this Sec-shRNA (e.g.,
primers, promoters, recognition RNA hairpin from Table II, shRNA,
and Pol III poly-T termination sequence) is released from the
pEGENSR plasmid with appropriate restriction enzymes and ligated
into the pEGEN FP vector comprising the fusion protein to create
the final plasmid pBioR(1).
[0311] Specific examples of various Sec-shRNAs delivered by various
CPP-RBD fusion proteins are shown in Table I and further described
in U.S. Ser. No. 61/160,287 and 61/160,288 (Examples 1-20), both of
which are incorporated by reference herein in their entireties.
[0312] Also, a different biologically active RNA sequence, such as
an antisense, ribozyme, aptamer, allozyme, siRNA, miRNA, or any of
the other biologically active molecules described herein, can be
used to substitute the shRNA sequence in the described pEGENSR
vector.
Example 3
Construction of the Bioreactor Plasmid pBioR(2) with a Sec-shRNA
Delivered by a CPP-NCS-RBD Fusion Protein
[0313] Delivery of Sec-shRNAs targeting any of the gene targets
from Table I and Table VII, as well as any other gene targets, is
also accomplished with the plasmid pBioR(2), which is constructed
using the same methods described in Examples 1 and 2. pBioR 2
encodes a fusion protein comprising a non-classical secretory
domain from Table V fused to an RNA binding domain from Table III
and a cell penetrating peptide from Table IV. This fusion protein
is assembled with or without alpha helical linker or other linker
domains. The expression cassettes for the fusion protein and the
Sec-shRNA are ligated into the pEGEN plasmids from Table VIII using
the methods described in Examples 1 and 2. Specific examples of
various Sec-shRNAs delivered by various CPP-NCS-RBD fusion proteins
are shown in Table I and further described in U.S. Ser. No.
61/160,287 and 61/160,288 (Examples 21-26), both of which are
incorporated by reference herein in their entireties.
Example 4
Construction of the Bioreactor Plasmid pBioR(3) with a Sec-shRNA
Delivered by a CPP-NCS-RBD Fusion Protein
[0314] Delivery of Sec-shRNAs targeting any of the gene targets
from Table I and Table VII, as well as any other gene targets, is
also accomplished with the plasmid pBioR(3). The plasmid pBioR(3)
is constructed using the same methods described in Example 1 for
the construction of pBioR(1) and is similar to pBioR(2) except that
it contains an additional expression cassette encoding an shRNA
molecule targeting the Dicer protein of the bioreactor cell. The
shRNA targeting Dicer has the following sequence:
TTGGCTTCCTCCTGGTTATGTTCAAGAGACATAACCAGGAGGAAGCCAA. The expression
cassettes for the fusion protein and the Sec-shRNA are ligated into
the pEGEN plasmids from Table VIII using the methods described in
Examples 1 and 2. The shRNA targeting Dicer is expressed from the
human H1 promoter and ends with a Pol-III poly-T terminator. An
example of a plasmid having an additional cassette encoding an
shRNA molecule targeting the Dicer protein is shown in FIG. 13.
Example 5
Construction of the Bioreactor Plasmid pBioR(14) with a Sec-shRNA
Delivered by a NCS-RBD-CPP Fusion Protein
[0315] Delivery of Sec-shRNAs targeting any of the gene targets
from Table I and Table VII, as well as any other gene targets, is
accomplished with the plasmid pBioR(14). The plasmid pBioR(14) is
constructed using the same methods described in Examples 1 and 2
for the construction of pBioR(1) and is similar to pBioR(2) except
for the location of the expression cassette for the Sec-shRNA. The
Sec-shRNA accompanies a fusion protein comprising a non-classical
secretory domain from Table V fused to an RNA binding domain from
Table III and a cell penetrating peptide from Table IV. In this
plasmid, the Sec-shRNA is encoded within an artificial intron
placed in either the 5' untranslated region (UTR) or within the
coding sequence for the fusion protein. The Sec-shRNA sequence is
subcloned between the splice donor and splice acceptor sites of the
artificial intron using appropriate restriction sites. This
multifunctional transcript is expressed from the chicken
.beta.-actin promoter and terminates with a human growth hormone
polyadenylation signal. Examples of plasmids having this type of
construction are shown in FIGS. 11 and 12.
Example 6
Construction of the Bioreactor Plasmid pBioR(15) with a
Sec-Ribozyme Delivered by a NCS-RBD-CPP Fusion Protein
[0316] Delivery of Sec-ribozymes targeting any of the gene targets
listed in Table I and Table VII, as well as any other gene targets,
is accomplished with the plasmid pBioR(15), constructed using the
same methods described in Examples 1 and 2 for the construction of
pBioR(1) encoding a fusion protein comprising a non-classical
secretory domain from Table V fused to an RNA binding domain from
Table III and a cell penetrating peptide from Table IV. The
Sec-ribozyme that accompanies this particular fusion protein
comprises an RNA recognition element from Table II and a RNA
ribozyme that targets any of the mRNA transcripts of the gene
targets listed in Table I and Table VII. The expression cassettes
for the fusion protein and the Sec-ribozyme are ligated into the
pEGEN2.1 plasmid. The fusion protein is expressed from the chicken
.beta.-actin promoter and terminates with a human growth hormone
polyadenylation signal and the Sec-Ribozyme is expressed from the
human U6 promoter and ends with a Pol-III poly-T terminator.
Specific examples of expression vectors for Sec-ribozyme RNAs and
CPP-NCS-RBD fusion proteins are described in U.S. Ser. No.
61/160,287 and 61/160,288 (Examples 39 and 40), both of which are
incorporated by reference herein in their entireties.
Example 7
Construction of the Bioreactor Plasmid pBioR(16) with a
Sec-Antisense RNA (Sec-asRNA) Delivered by an NCS-RBD-CPP Fusion
Protein
[0317] Delivery of Sec-asRNAs targeting any of the gene targets
listed in Table I and Table VI, as well as any other gene targets,
is accomplished with the plasmid pBioR(16), constructed using the
same methods described in Examples 1 and 2 for the construction of
pBioR(1) encoding a fusion protein comprising a non-classical
secretory domain from Table V fused to an RNA binding domain from
Table III and a cell penetrating peptide from Table IV. The
Sec-asRNA that accompanies this particular fusion protein comprises
an RNA recognition element from Table II and an antisense RNA
complementary to any of the mRNA transcripts of gene targets listed
in Table I and Table VII. The expression cassette for the fusion
protein is ligated into the pEGEN2.1 plasmid and is expressed from
the chicken .beta.-actin promoter and terminates with a human
growth hormone polyadenylation signal. The expression cassette for
the Sec-asRNA is ligated into the pEGEN1.1 plasmid and is expressed
from the SV40 promoter and terminates with a human growth hormone
polyadenylation signal. The expression cassette for this Sec-asRNA
(primers, U6 promoter, recognition RNA hairpin from Table II,
asRNA, and Pol III poly-T termination sequence) is released from
the pEGEN1.1 plasmid with appropriate restriction enzymes and
ligated into the pEGEN 2.1/FP vector comprising the fusion protein
to create the final plasmid pBioR(16) as described in Example 2.
Specific examples of expression vectors for Sec-asRNAs and
CPP-NCS-RBD fusion proteins are described in U.S. Ser. No.
61/160,287 and 61/160,288 (Examples 41 and 42), both of which are
incorporated by reference herein in their entireties.
Example 8
Construction of the Bioreactor Plasmid pBioR(17) with a
Sec-Antisense RNA (Sec-asRNA) Delivered by an NCS-RBD-CPP Fusion
Protein
[0318] Delivery of Sec-asRNAs targeting any of the gene targets
from Table I and Table VII, as well as any other gene targets, is
accomplished with the plasmid pBioR(17), constructed using the same
methods described in Examples 1 and 2 for the construction of
pBioR(1) encoding a fusion protein comprising a non-classical
secretory domain from Table V fused to an RNA binding domain from
Table III and a cell penetrating peptide from Table IV. The
Sec-asRNA that accompanies this particular fusion protein comprises
an RNA recognition element from Table II and an antisense RNA
complementary to any of the mRNA transcripts of the gene targets
listed in Table I and Table VII. The expression cassette for the
fusion protein and the Sec-asRNA are ligated into the pEGEN2.1
plasmid and is expressed from the chicken .beta.-actin promoter and
terminated with a human growth hormone polyadenylation signal. In
this plasmid, the Sec-asRNA is encoded within an artificial intron
placed either in the 5' untranslated region (UTR) or within the
coding sequence for the fusion protein. This multifunctional
transcript is expressed from the chicken .beta.-actin promoter and
terminates with a human growth hormone polyadenylation signal.
Example 9
Construction of the Bioreactor Plasmid pBioR(18) with a Sec-Aptamer
Secreted by a NCS-RBD Fusion Protein
[0319] Delivery of Sec-aptamer targeting extracellular receptor
proteins listed in Table I and Table VII, as well as any other
extracellular receptor proteins, is accomplished with the plasmid
pBioR(18), constructed using the same methods described in Examples
1 and 2 for the construction of pBioR(1), encoding a fusion protein
comprising a non-classical secretory domain from Table V fused to
an RNA binding domain from Table III. The Sec-aptamer that
accompanies this particular fusion protein comprises an RNA
recognition element from Table II and an aptamer sequence that
targets any of the extracellular receptor proteins listed in Table
I and Table VII. The expression cassettes for the fusion protein
and the Sec-aptamer are ligated into the pEGEN2.1 plasmid. The
fusion protein is expressed from the chicken .beta.-actin promoter
and terminates with a human growth hormone polyadenylation signal
and the Sec-aptamer is expressed from the human U6 promoter and
ends with a Pol-III poly-T terminator.
[0320] Specific examples of expression vectors for Sec-Aptamers and
NCS-RBD fusion proteins are described in U.S. Ser. No. 61/160,287
and 61/160,288 (Examples 43 and 44), both of which are incorporated
by reference herein in their entireties.
Example 10
Construction of the Bioreactor Plasmid pBioR(19) with a Sec-Aptamer
Secreted by a NCS-RBD-CPP Fusion Protein
[0321] Delivery of Sec-aptamer targeting any of the cellular
proteins listed in Table I and Table VII, as well as any other
cellular proteins, is accomplished with the plasmid pBioR(19),
constructed using the same methods described in Examples 1 and 2
for the construction of pBioR(1) encoding a fusion protein
comprising a non-classical secretory domain from Table V fused to
an RNA binding domain from Table III and a cell penetrating peptide
from Table IV. The Sec-aptamer that accompanies this particular
fusion protein comprises an RNA recognition element from Table II
and an aptamer sequence that targets any of the intracellular
proteins listed in Table I and Table VII. The fusion protein and
Sec-aptamer are constructed the same way and are expressed from the
same promoters as described in Examples 1 and 2 for pBioR(1).
[0322] Specific examples of expression vectors for Sec-Aptamers and
CPP-NCS-RBD fusion proteins are described in U.S. Ser. No.
61/160,287 and 61/160,288 (Examples 45 and 46), both of which are
incorporated by reference herein in their entireties.
Example 11
Administration of Bioreactor Plasmids to HeLa Cells in Culture
Using Polymer Mediated Transfection
[0323] Bioreactor cells are generated by co-transfecting pEGENFP
and pEGENSR (see Example 2) into a recipient cell line, for example
HeLa cells, in vitro. HeLa cells are cultured in six-well plates in
DMEM+10% fetal bovine serum (2 mL total volume) to a density of 80%
confluence in preparation for transfection by a polymeric delivery
agent. Growth media is removed from the cells and replaced with 1
mL of DMEM only (no serum) preheated to 37.degree. C. Transfection
complexes are formed between the delivery reagent and the pBioR
plasmid by incubation in DMEM at room temperature for 20 minutes
(DNA and reagent concentrations optimized for each application).
Transfection complexes are added to the HeLa cells by dropwise
addition to the each culture and returned to the 37.degree. C.
incubator. After a five hour incubation, DMEM+20% serum is added to
the transfection media to produce a final concentration of 10%
serum and a final volume of 2 mL. Transiently transfected cells are
ready for use as BioReactors by addition to target cells.
Example 12
Administration of Bioreactor Plasmid to Cells in Culture Using
Polymer Mediated Transfection
[0324] BioReactor cells are generated by transfecting a pBioR
plasmid (any plasmid described elsewherein the application and in
the previous examples) into a recipient cell line in vitro.
Transfection protocols for generation of transiently transfected
BioReactor cells are similar to those described in Example 11 for
the generation of BioReactors based on HeLa cells. Non-limiting
examples of suitable recipient cells in culture include A549 cells,
Jurkat cells, HepG2 cells, NIH3T3 cells, Renka cells, CT26 cells,
PC-12 cells, Cos-1 cells, Cos-7 cells, and CHO cells. The methods
described in Example 11 can be applied to these cells in culture,
as well as to other known established cell lines.
Example 13
Administration of Bioreactor Plasmid to HeLa Cells in Culture Using
Electroporation Mediated Transfection
[0325] BioReactor cells are produced from HeLa recipient cells by
transfection with the pBioR plasmid by electroporation. HeLa cells
are cultured in 100 mm culture dishes in DMEM+10% fetal calf serum
(15 mL total volume) to a density of 80% confluence in preparation
for electroporation. Cells are released from the wells with trypsin
and collected by centrifugation (500.times.g for 5 minutes at
4.degree. C.). The cell pellet is resuspended in growth medium and
the cell density is measured using a hemocytometer; the final
volume is adjusted with growth medium to yield 5.times.10.sup.6
cells/mL. The cells are transferred to the electroporation cuvette
along with 20 ug of the pBioR plasmid and placed in between the
electrodes. The electroporator is discharged at 260V
(Capacitance=1000 .mu.F, infinite internal resistance) and the
cuvette is allowed to rest for 2 minutes. Electroporated cells are
then transferred to a culture dish along with two rinses of the
cuvette with growth medium. Cells are grown at 37.degree. C. under
5% CO.sub.2 for 48 hours.
[0326] BioReactor cells are produced from other recipient cells by
transfection with the pBioR plasmid as described above for the
generation of BioReactors based on HeLa cells. Non-limiting
examples of suitable recipient cells in culture include A549 cells,
Jurkat cells, HepG2 cells, NIH3T3 cells, Renka cells, CT26 cells,
PC-12 cells, Cos-1 cells, Cos-7 cells, and CHO cells. Assays that
demonstrate function of the BioReactor cell are as described in
Example 16.
Example 14
Administration of Bioreactor Plasmid to HeLa Cells in Culture Using
Viral Mediated Transfection
[0327] Viral vectors are constructed from isolated plasmid
backbones, expression cassettes for the structural and
non-structural components of the virus and expression cassettes for
the biologically active RNA. PCR amplification of expression
cassettes, subcloning of expression cassettes into plasmid
backbones, amplification and isolation of the resulting virus
producing vectors and subsequent verification of plasmid sequences
are all carried out as described in Example 1. Viral vectors are
constructed from one of several DNA viral expression cassettes such
as Adenovirus and Adeno-associated virus (2-3, 7, 11, 19, 21) and
Herpes Simplex Virus (5, 14-15, 18) or RNA viral expression
cassettes such as Lentivirus (6, 20, 22, 24), Sindbis Virus (9),
Murine Leukemia Virus (10.mu., 12-13, 16) or Foamy Virus (8, 17)
and any of the biologically active RNA molecules described
elsewhere in the application and in the previous examples. For each
virus, the structural genes encoding viral coat proteins and
fusogenic proteins are subcloned into any of the pEGEN backbone
plasmids for expression from a Pol-II promoter sequence generating
pVir1. Separately, the non-structural genes encoding the
polymerases and accessory proteins are coupled with the
biologically active RNA sequence and fusion protein sequence and
subcloned into a second pEGEN plasmid for expression from a Pol-II
promoter sequence generating pVir2. Plasmids pVir1 and pVir2 are
co-transfected into recipient cells to generate virus producing
cells. Virus particles can then be purified and concentrated for
use in administration of the bioreactor expression cassettes to
bioreactor cells.
Example 15
Administration of Bioreactor Plasmid to HeLa Cells in Culture Using
Polymer Mediated Transfection and Generation of Stable Cell
Lines
[0328] BioReactor cells are produced from HeLa recipient cells by
transfection with the pBioR plasmid as described in Examples 11-14.
Stable integration of the pBioR plasmid into the recipient cell
genome is achieved by extended growth in selective media. pBioR
plasmids for stable integration contain a puromycin resistance gene
or a G418/Neomycin resistance gene in addition to the pUC origin
and kanamycin resistance gene. Newly transfected cells are allowed
to recover in complete, non-selective media for 48 hours. These
cells are then transferred to selective media and grown at
37.degree. C. under 5% CO2 with media changes every 3 days.
Individual isolates of cells with stably integrated plasmids are
moved to individual wells and expanded. These expanded cell lines
are then assayed for optimal bioreactor activity. Assays that
demonstrate function of the BioReactor cell are as described in
Example 16.
Example 16
Assays for Confirming the Production and Secretion of the
RNA-Protein Complex in Cell Culture
[0329] Cells are transfected with a pBioR expression vector or a
null vector using the methods described in Examples 11-14.
Successful generation of BioReactor cells is confirmed by assays
that verify one or more of the following: (1) production of the
fusion protein, (2) production of the Sec-RNA, (3) binding of the
Sec-RNA by the fusion protein and (4) successful secretion of the
RNA-protein complex. Production of the fusion protein can be
verified through RT-PCR based assays that detect the plasmid
derived mRNA transcript encoding the fusion protein and antibody
based assays that detect the fusion protein itself. For purposes of
detecting the fusion protein, short "protein tags" which are
recognized by commercially available antibodies, can be included in
the sequence of the fusion protein. These protein tags are used to
verify the function of the BioReactor cell and are not necessarily
included in the functional BioReactor fusion proteins.
[0330] To detect the plasmid derived mRNA transcript, total RNA is
prepared from pBioR-transfected, null vector-transfected, and
non-transfected cells, i.e., HeLa cells or any of the other cells
described herein and otherwise known in the art, using Tri-Reagent
(Sigma-Aldrich, product #T9424) according to the manufacturer's
protocols. A cDNA library is prepared from the total RNA using a
poly-T primer and used as template for the PCR amplification.
Primers for two separate amplification reactions, each producing a
different size product, are included in the PCR reactions: (1)
Primers amplifying sequences from an internal control gene, such as
.beta.-actin or GAPDH, and (2) Primers amplifying sequences
specific to the mRNA encoding the fusion protein. Products are
resolved on 2% agarose gels run in 1.times.TAE or on 10% acrylamide
gels run in 1.times.TBE. Products are compared for the
non-transfected cells (negative control), cells transfected with a
null vector (backbone vector without the fusion protein), and the
potential BioReactors (i.e., cells transfected with a pBioR)
through staining with ethidium bromide and illumination with UV
light at 302 nm. Non-transfected control cells have a single PCR
product for the internal control gene while successful BioReactors
have products for both the internal control gene and the transcript
encoding the fusion protein.
[0331] Direct detection of the fusion protein is accomplished by
collection of total protein from pBioR-transfected, null
vector-transfected, and non-transfected cells, as well as the media
in which those cells are growing. Total protein is concentrated
from each sample by acetone precipitation and the concentrated
proteins are resuspended in either a native buffer for ELISA
analysis or denaturing buffer for western blot analysis. Each assay
utilizes standard methods and antibodies specific for an internal
control gene (.beta.-actin or GAPDH) and a protein tag present in
the fusion protein. As discussed, protein tags are included in the
fusion proteins as a convenient means for verifying function of the
BioReactor cell. Non-transfected and null vector-transfected
control cells have a single protein detected for the internal
control gene while successful BioReactors have both the internal
control protein and the fusion protein.
[0332] Successful production of the Sec-RNA includes both
transcription of the RNA and export of that transcript from the
nucleus. RT-PCR assays are used to show production of the plasmid
derived Sec-RNA molecule and cellular fractionation is used to
demonstrate accumulation of the RNA in the cytoplasm. The cellular
fractionation is accomplished with the PARIS RNA isolation kit
(Ambion, Product #1921) according to the manufacturer's protocol. A
cDNA library is prepared from the fractionated RNA using a random
hexamer non-specific primer and is used as template for the PCR
amplification. Primers for two separate amplification reactions,
each producing a different size product, are included in the PCR
reactions: (1) Primers amplifying sequences from an internal
control gene, such as .beta.-actin or GAPDH, and (2) Primers
amplifying sequences specific to the Sec-RNA. Products are resolved
on 2% agarose gels run in 1.times.TAE or on 10% acrylamide gels run
in 1.times.TBE. Products are compared for the null
vector-transfected and non-transfected cells (negative controls)
and the potential BioReactors through staining with ethidium
bromide and illumination with UV light at 302 nm. Null
vector-transfected and non-transfected control cells have a single
PCR product for the internal control gene while successful
BioReactors have products for both the internal control gene and
the Sec-RNA.
[0333] FIG. 16 shows the results of experiments to confirm the
expression of Sec-RNA and the fusion protein. For the secreted RNA
reporter transcript analyses shown in FIG. 16A, CT26 cells were
transfected with pE3.1 Sec-Reporter (FIG. 15A). After 48 hours,
total cellular RNA was collected from untransfected control cells
and transfected bioreactor cells using Quigen's RNEasy kit
according to the manufacturer's recommended protocol and purified
RNA was amplified using RT-PCR and separated on 3% low melt agarose
gels (1.times.TAE). RT-PCR reactions for the sec-RNA included
probes and primers for amplifying both 18S rRNA (internal control,
196 bp product) and the secreted RNA reporter (100 bp product).
Untransfected control cells ("U") show only the 18S rRNA internal
control (18S) whereas the transfected cells show both the 18S rRNA
product and the parent reporter RNA product ("R"), which
corresponds to the plasmid only, or the secreted reporter RNA
product ("SR"), which corresponds to the plasmid and the Sec-RNA
sequence insert. FIG. 16B shows the fusion protein expression
analyses, in which CT26 cells were transfected with plasmids
expressing the bioreactor fusion protein. After 48 hours, the cells
were harvested in TENT buffer, boiled for 5 minutes, spun at
16,000.times.G for 15 minutes to remove the cellular debris and
allow for collection of the cell lysate (total protein). Aliquots
of cell lysates from untransfected cells and cells transfected with
pE3.1 Sec-Reporter and either pE1.1 TAT+(TAT fused to a Protein N
RNA binding domain and 6.times. Histidine epitope tag) or
pE2.1TAT+(TAT fused to a Protein N RNA binding domain and 6.times.
Histidine epitope tag) were spotted to PVDF membranes along with a
positive control protein for the blotting antibody. The blots were
developed with chromogenic substrates and recorded with an image
documentation center.
[0334] Binding of the Sec-RNA molecule by the fusion protein is
demonstrated by immunoprecipitation of the RNA-protein complex via
the peptide tags described above. Antibodies specific for an
internal control gene (.beta.-actin or GAPDH) or the protein tag
present in the fusion protein are coupled to protein-A sepharose
(PAS) beads or protein-G sepharose (PGS) beads. Beads are
rehydrated in cell lysis buffer and antibodies are coupled by
incubation with beads at 4.degree. C. overnight. A non-specific
antibody, often a preimmune serum, is used as a negative control
for the immunoprecipitation assay. The antibody coupled beads are
spun out of solution (1500.times.g for 5 minutes), the supernatant
is removed, and the antibody coupled beads are washed with cell
lysis buffer. Proteins are prepared from pBioR-transfected, null
vector-transfected, and non-transfected cells, as well as the media
in which those cells are growing The proteins are collected in
native cell lysis buffers in order to preserve the RNA-protein
complexes, the precise composition of which is adjusted to the
specific purification. A typical cell lysis buffer composition is
20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, and 0.05% Nonidet
P-40. Protein extracts are added to the antibody coupled beads and
the immunoprecipitation is carried out under conditions optimized
for each reaction. Typical precipitations are incubated at room
temperature for 2 to 4 hours. Isolated RNA-protein complexes are
spun out of solution and the supernatant is collected as the
precipitation input. The beads are washed repeatedly to remove
non-specifically bound proteins; the total number of washes is
empirically determined for each precipitation. Isolated complexes
are eluted from the beads with a peptide matching that of the
fusion protein tag which competes for the binding sites present on
the antibody. Isolated RNAs are then detected by northern blotting
or by RT-PCR as described above.
[0335] Successful secretion of the RNA-protein complex is verified
by detection of the Sec-RNA in the extracellular matrix, or media
in the case of cells in culture. Intact RNA-protein complexes may
be isolated from the media via immunoprecipitation, as described
above, or total RNA may be prepped using Tri-Reagent in accordance
with the manufacturer's protocol (Sigma-Aldrich, product #T9424).
The Sec-RNA is detected by northern blotting or by RT-PCR as
described above.
[0336] FIG. 17 shows the results of an experiment to confirm the
secretion of an RNA-protein complex from a bioreactor cell. Total
cellular RNA from untransfected control CT26 cells and CT26 cells
transfected with the pE3.1 Sec-Reporter and pE1TAT-RBD plasmids
expressing the secreted RNAs and the bioreactor fusion proteins was
collected after 48 hours transfection using Qiagen's RNEasy kit
according to the manufacturer's recommended protocol. RNA was also
collected from the cell culture media and purified using the
RNAeasy kit. The purified RNA was used as template for RT-PCR
amplification reactions and the amplified products were separated
on 3% low melt agarose gels (1.times.TAE) along with DNA size
standards. RT-PCR was carried out with probes and primers for both
18S rRNA (internal control) and the secreted RNA reporter. FIGS.
17A and 17B show the results of a transfection assay with pE3.1
Sec-Reporter and either pE1.1 TAT(+) (TAT fused to the proper RBD)
or pE1.1 TAT(-) (TAT fused to a negative control RBD). The left
hand panel of FIG. 17A shows RT-PCR products for cell lysates
collected from cells transfected with the parent reporter plasmid
("R"), the reporter plasmid containing the sec-RNA sequence insert
("SR"), the sec-RNA reporter plasmid co-transfected with pE1.1
TAT(+) or with pE1.1 TAT(-). The right hand panel of FIG. 17A shows
both cell lysates ("C") and extracellular media samples ("M") from
cells cotransfected with the sec-RNA reporter plasmid and pE1.1
TAT(+) or pE1.1 TAT(-). As shown, in cells transfected with the
pE3.1 Sec-Reporter and pE1TAT(+) plasmids, the RNA-protein complex
is secreted into the media, whereas in cells transfected with the
pE3.1 Sec-Reporter and pE1TAT(-) plasmids (TAT fused to a negative
RBD control), the fusion protein (sec-RNA) was not present in the
media.
Example 17
Assaying CPP-Mediated Secretion Activity of a Luciferase/Alkaline
Phosphatase Reporter Gene
[0337] FIG. 14A is a non-limiting schematic showing an exemplary
transfection assay to generate and test the secretory activity of
bioreactor cells using the CPP-Luciferase/CPP-Alkaline Phosphatase
reporter system. Fusion protein cassettes fusing cell penetrating
peptides to a luciferase reporter gene are generated via PCR. These
PCR products include restriction sites at each end of the DNA to
facilitate subcloning into the pEGEN1.1 plasmid, placing the fusion
protein cassette between an SV40 promoter and an hGH poly-A tail
sequence. The resulting plasmids are transfected into a number of
different cell types in vitro to generate BioReactor reporter cells
as described in Examples 11-14. Total protein is collected from the
growth media and the cells and luciferase activity is measured in
both to establish the distribution of tagged luciferase molecules
inside and outside the cell. Requirements for secretion are
established through comparison to control proteins including
luciferase/alkaline phosphatase alone and luciferase/alkaline
phosphatase fused to a scrambled CPP domain.
[0338] FIG. 14B shows CPP-mediated secretion of the luciferase
reporter protein from cells transfected with reporter plasmids and
cultured in vitro. CT26 cells were transfected with plasmids
expressing luciferase or a TAT-luciferase fusion protein. After 48
hours, cell media was replaced with PBS and cells were incubated at
37.degree. C. for an additional 3 hours. The PBS supernatant was
collected and the cells were lysed in TENT buffer. Luciferase
activity was measured for equivalent amounts of solubilized
cellular protein and PBS supernatant using standard methods. The
relative luciferase activity present in cellular and supernatant
fractions is presented as a percentage of the total luciferase
activity observed in both fractions. The addition of the TAT cell
penetrating peptide to the luciferase reporter protein shifts the
distribution of luciferase activity out of the transfected cell and
into the supernatant.
Example 18
Assaying CPP-Mediated Delivery of a Split GFP Reporter Gene
[0339] FIG. 18 is a non-limiting schematic showing an exemplary
transfection assay to generate and test the import activity of
bioreactor cells using the GFP reporter system. Fusion protein
cassettes fusing cell penetrating peptides to an isolated domain
from a split GFP reporter system are generated by PCR. A separate
PCR reaction generates a protein cassette encoding a GFP
complementary fragment. These PCR products each include restriction
sites at each end of the DNA to facilitate subcloning into the
pEGEN1.1 plasmid, placing the fusion protein cassette and the GFP
complimentary fragment cassette between an SV40 promoter and an hGH
poly-A tail sequence. The resulting plasmids are transfected
independently into cells in vitro to generate Bioreactor reporter
cells expressing the CPP-GFP fusion protein and target cells
expressing the GFP complimentary fragment. The experiment is
initiated by mixing the bioreactor cells with the target cells.
Secretion of the CPP fusion protein from the bioreactor cells and
subsequent import into the target cells will be detected upon
docking of the activating domain to the GFP complimentary fragment
by the resulting GFP signal.
Example 19
Application of the Bioreactor Cell Transfection Reagent to HeLa
Cells for the Purpose of mRNA Transcript Knockdown in Culture
[0340] Bioreactor cells, such as those produced from Examples 11-14
and confirmed using the methods described in Examples 16, are
applied directly to target cells for the purpose of knocking down
the gene product targeted by the Sec-RNA molecule. The particular
pBioR plasmid and recipient cells used in the transfection are
determined by the gene target of interest and the target cell
identity. In this example, the HeLa target cells are transfected
with NIH3T3 BioReactor cells secreting a Sec-shRNA-fusion protein
complex with an shRNA targeting the VEGF transcript. In using mouse
derived BioReactor cells to transfect human derived target cells,
it is possible to observe knockdown of the VEGF transcript in the
human target cells through the use of species specific primer sets.
Depletion of VEGF protein in human cells and subsequent decreases
in the amount of secreted protein can also be detected in the media
using assays with VEGF antibodies specific for the human
protein.
[0341] BioReactor cells are produced from NIH3T3 recipient cells by
transfection of NIH3T3 cells with the pBioR plasmid as described in
Examples 11-14. BioReactor function is also verified with assays
described in Example 16. It is not necessary to separate or purify
the BioReactor cells following transient transfection of the NIH3T3
cells. HeLa cells are cultured in 6 well plates in DMEM+10% fetal
bovine serum (2 mL total volume) to a density of 50% confluence.
BioReactor cells are collected by trypsinization and centrifugation
(500.times.g for 5 minutes). The cell pellet is resuspended in the
same growth medium used for the HeLa target cells and the cell
density is measured using a hemocytometer. Bioreactor cells are
added to the HeLa target cells and the combined culture is
incubated at 37.degree. C. under 5% CO.sub.2. The optimal ratio of
BioReactor cells to target cells is determined empirically for each
system of cells and gene targets. RNA or protein samples are
collected from each cell culture 48-96 hours after addition of the
BioReactor cells in order to assay knockdown of the mRNA transcript
or protein, respectively, as described in Example 16.
Example 20
Bioreactor Mediated Delivery of an RNA Aptamer to the Extracellular
Matrix
[0342] This example describes an exemplary transfection assay to
determine the secretion activity of bioreactor cells secreting an
aptamer, for example, an aptamer targeted to Oncostatin M protein,
which is an activator of the gp130 receptor mediated signaling
pathway (see FIG. 19). The assay employs the use of a reporter
system and a secreted RNA aptamer targeting the Oncostatin M
protein. An expression plasmid for the fusion protein (pEGENFP,
Example 2) and as expression plasmid for an RNA aptamer (pEGENSR,
Example 2) targeting Oncostatin M are transfected into a number of
different cell types in vitro to generate Bioreactor cells
secreting the RNA aptamer as described in Examples 11-14. A
reporter plasmid expressing the luciferase protein under the
control of promoter elements responsive to the gp130 mediated STAT3
signaling pathway (SABiosciences, Cignal Reporter Assays, Catalog
#CCS-9028) is transfected into HepG2 cells (gp130 expressing cells)
in vitro to generate target (reporter) cells. After 48 hours, cell
media is collected from the bioreactor cells secreting the aptamer
for Oncostatin M and incubated with a recombinant Oncostatin M
protein (0.2-20 ng/mL) for 3 hours at room temperature to allow for
binding of the secreted aptamer to the target protein. The media is
then transferred to the target (reporter) cells and cultures are
incubated at 37.degree. C. for 24 hours. Controls include addition
of recombinant Oncostatin M protein directly to reporter cells,
Oncostatin M incubated with media from untransfected cells,
Oncostatin M incubated with media from bioreactor cells transfected
with only the RNA aptamer expressing plasmid (pEGENSR), Oncostatin
M incubated with media from cells expressing mismatched RNA binding
domains and Oncostatin M treated with RNA aptamers purified from
pEGENSR transfected cells. Luciferase assays are carried out as
described in Example 17. Reporter cells incubated with the media
containing the aptamer targeting Oncostatin M will have less
luciferase activity than reporter cells incubated with Oncostatin M
alone or incubated with Oncostatin M and control media. The
secretion of other aptamers from bioreactor cells can be assayed
using similar methods with the appropriate luciferase or other
reporter vector system.
Example 21
Bioreactor Mediated Delivery of an RNA Aptamer to the Extracellular
Matrix
[0343] This example describes an exemplary transfection assay to
determine the secretion activity of bioreactor cells secreting an
aptamer, for example, an aptamer targeted to HER3 (see FIG. 20).
The assay employs the use of a reporter system and a secreted RNA
aptamer targeting the HER3 protein. An expression plasmid for the
fusion protein (pEGENFP, Example 2) and as expression plasmid for
an RNA aptamer (pEGENSR, Example 2) targeting HER3 are transfected
into a number of different cell types in vitro to generate
Bioreactor cells secreting the RNA aptamer as described in Examples
11-14. Reporter cells expressing the HER3 receptor protein (MCF7
for example) are cultured separately. After 48 hours, cell media is
collected from the bioreactor cells secreting the aptamer for HER3
and transferred to the HER3 expressing reporter cells and cultures
are incubated at 37.degree. C. for 24-72 hours. Controls include
addition of media from untransfected cells, media from bioreactor
cells transfected with only the RNA aptamer expressing plasmid
(pEGENSR), media from cells expressing mismatched RNA binding
domains and with RNA aptamers purified from pEGENSR transfected
cells. Cell growth is monitored using Promega's CellTiter 96
Aqueous Non-Radioactive Cell Proliferation Assay (Catalog #G5421)
according to the manufacturer's protocol. Reporter cells incubated
with the media containing the aptamer targeting HER3 will show less
cell growth than reporter cells incubated with control media. The
secretion of other aptamers from bioreactor cells can be assayed
using similar methods with the appropriate reporter vector
system.
Example 22
Bioreactor Mediated Delivery of an shRNA to the Cytoplasm of a
Target Cell
[0344] This example describes an exemplary transfection assay to
determine the secretion activity of bioreactor cells and subsequent
delivery of an inhibitory shRNA to the cytoplasm of a target cell
(see FIG. 21). An expression plasmid for the fusion protein
(pEGENFP, example 2) and an expression plasmid for the shRNA
(pEGENSR, example 2) are transfected into a number of different
cell types in vitro to generate Bioreactor cells as described in
Examples 11-14. Target cells expressing the mRNA transcript
targeted by the shRNA are cultured separately. After 48 hours, cell
media is collected from the bioreactor cells and transferred to the
target cells and cultures are incubated at 37.degree. C. for 24-72
hours. Controls include addition of media from untransfected cells,
media from bioreactor cells transfected with only the shRNA
expressing plasmid (pEGENSR), media from cells expressing
mismatched RNA binding domains and with shRNAs purified from
pEGENSR transfected cells. Total RNA is prepared from the target
cells and RT-PCR analysis is carried out as described in Example
11. Knockdown of the target gene is assessed by comparison to a
non-targeted internal control gene. Alternatively, bioreactor cells
and target cells can be cultured together during the experiment if
the primers and probes used in the RT-PCR assays do not recognize
the corresponding transcripts in the bioreactor cells. This is most
easily achieved by using cell lines derived from one species for
bioreactor cells and cell lines derived from a different species
for the target cells. In this case, bioreactor cells can be
collected 24 hours after transfection and mixed with target cells
for direct assays of bioreactor activity as assayed by RT-PCR
analysis. Target cells expressing the mRNA transcript targeted by
the shRNA are cultured separately. The secretion of other shRNAs
from bioreactor cells can be assayed using similar methods with the
appropriate target cells.
Example 23
Ex Vivo Administration of the pBioR Expression Vectors to Cells
[0345] BioReactor cells are produced from NIH3T3 recipient cells by
transfection with the pBioR plasmid as described in Examples 11-14.
BioReactor function is verified with assays described in Example
16. In this example, the NIH3T3 BioReactor cells secrete an
Sec-shRNA-fusion protein complex with an shRNA targeting the VEGF
transcript. The BioReactor cells are mixed with SCCVII target cells
(a mouse squamous cell carcinoma line) and the mixture is
transplanted into nude mice (immune-compromised) by subcutaneous
injection into the rear flanks of each animal. BioReactor activity
is monitored by assessment of VEGF transcript and protein levels in
tissues surrounding the transplantation site compared with
controls. Bioreactor function are also be assessed in vivo by
comparing tumor growth in the BioReactor/SCCVII transplants to
control mice receiving SCCVII cells alone or SCCVII cells with
non-functional BioReactor cells (non-specific shRNAs or delivery
compromised fusion proteins).
Example 24
In Vivo Administration of BioReactor Cells to Mouse Muscle
Tissue
[0346] BioReactor cells are produced from primary mouse myoblast
recipient cells by transfection with the pBioR plasmid as described
in Examples 11-14. BioReactor function is verified using assays
described in Examples 16. In this example, BioReactors cells
secrete an Sec-shRNA-fusion protein complex with an shRNAs
targeting the mRNA transcript for myostatin, a negative regulator
of skeletal muscle growth. The BioReactor cells are transplanted
into the tibialis muscle of mdx mice, a model system for Duchenne
muscular dystrophy (Li S, Kimura E, Ng R, Fall B M, Meuse L, Reyes
M, Faulkner J A, Chamberlain J S., A highly functional
mini-dystrophin/GFP fusion gene for cell and gene therapy studies
of Duchenne muscular dystrophy., Hum Mol Genet. 2006 May 15;
15(10):1610-22). BioReactor activity is monitored by assessment of
myostatin transcript and protein levels in tissues surrounding the
transplantation site. RNA and protein samples are prepared from
tibialis muscles collected from untreated mice, mice transplanted
with BioReactor cells secreting non-specific Sec-shRNAs and mice
transplanted with BioReactor cells secreting shRNAs targeting the
myostatin transcript using Tri-Reagent (Sigma-Aldrich, product
#T9424). Relative levels of myostatin transcript and protein can
then be assessed by RT-PCR or ELISA, respectively, as described in
Example 16. BioReactor function is also assessed in vivo by
comparing body mass, muscle mass, muscle size and muscle strength
in the BioReactor transplants relative to control mice receiving no
BioReactor cells or non-functional BioReactor cells (Bogdanovich S,
Krag T O, Barton E R, Morris L D, Whittemore L A, Ahima R S,
Khurana T S., Functional improvement of dystrophic muscle by
myostatin blockade., Nature. 2002 Nov. 28; 420(6914):418-21).
Example 25
In Vivo Administration of BioReactor Cells to Mouse Neural
Tissue
[0347] BioReactor cells are produced from mouse neural stem cells
(mNSC) by transfection with the pBioR plasmid as described in
Examples 11-14. BioReactor function is verified with assays
described in Examples 16. In this example, the mNSC BioReactor
cells secrete an Sec-shRNA-fusion protein complex with an shRNA
targeting the mRNA transcript for the CAG repeat expansion of the
mutant huntingtin (htt) protein. The BioReactor cells are
transplanted into the brain of mouse models for Huntington's
disease to evaluate the efficacy of BioReactor mediated knockdown
of the mRNA transcript for the mutant form of the htt protein. RNA
samples are prepared from mouse brain tissue collected from
untreated mice, mice transplanted with BioReactor cells secreting
non-specific Sec-shRNAs and mice transplanted with BioReactor cells
secreting shRNAs targeting the mutant huntingtin transcript using
Tri-Reagent (Sigma-Aldrich, product #T9424). Relative levels of
huntingtin transcript can then be assessed by RT-PCR as described
in Example 16. Mouse models for Huntington's disease also display
abnormal protein build-up in striatal tissues and abnormal gaits,
both of which may provide physiological readouts of BioReactor
activity. See Yang C R, Yu R K., Intracerebral transplantation of
neural stem cells combined with trehalose ingestion alleviates
pathology in a mouse model of Huntington's disease., J Neurosci
Res. 2008 Aug. 5; 87(1):26-33.; DiFiglia M, Sena-Esteves M, Chase
K, Sapp E, Pfister E, Sass M, Yoder J, Reeves P, Pandey R K, Rajeev
K G, Manoharan M, Sah D W, Zamore P D, Aronin N., Therapeutic
silencing of mutant huntingtin with siRNA attenuates striatal and
cortical neuropathology and behavioral deficits., Proc Natl Acad
Sci USA. 2007 Oct. 23; 104(43):17204-9.
Example 26
Administration of BioReactor Cells to Human Synovial Fluid
[0348] BioReactor cells are produced from human synovial
fibroblasts by transfection with the pBioR plasmid as described in
Examples 11-14. BioReactor function is verified with assays
described in Examples 16. In this example, the fibroblast
BioReactor cells secrete an Sec-shRNA-fusion protein complex with
an shRNA targeting the mRNA transcript for either the IL-1.beta.,
the IL-6 or the IL-18 proinflammatory cytokines. The transfected
cells are expanded for injection of transciently transfected cells
or generation of stable cells via selective growth with
antibiotics. The BioReactor cells are resuspended in 1.times.PBS
(without Ca.sup.2+ or Mg.sup.2+) and injected into the joints of
arthritis patients (Evans C H, Robbins P D, Ghivizzani S C, Wasko M
C, Tomaino M M, Kang R, Muzzonigro T A, Vogt M, Elder E M,
Whiteside T L, Watkins S C, Herndon J H., Gene transfer to human
joints: progress toward a gene therapy of arthritis., Proc Natl
Acad Sci USA. 2005 Jun. 14; 102(24):8698-703). Sec-shRNA-fusion
protein complexes produced by the fibroblast BioReactor cells will
be delivered to the interleukin producing monocytes to reduce the
amount of cytokine present in the synovial fluid. BioReactor
function is assessed by monitoring the amount of IL-1.alpha., IL-6,
IL-18 and TNF.alpha. protein present in the synovial fluid, as well
as physiological indications of the disease. (Khoury M, Escriou V,
Courties G, Galy A, Yao R, Largeau C, Scherman D, Jorgensen C,
Apparailly F., Efficient suppression of murine arthritis by
combined anticytokine small interfering RNA lipoplexes., Arthritis
Rheum. 2008 August; 58(8):2356-67).
Example 27
Construction of the Viral Vector
[0349] Viral vectors are constructed from isolated plasmid
backbones, expression cassettes for the structural and
non-structural components of the virus and expression cassettes for
the biologically active RNA. PCR amplification of expression
cassettes, subcloning of expression cassettes into plasmid
backbones, amplification and isolation of the resulting virus
producing vectors and subsequent verification of plasmid sequences
are all carried out as described in Example 1. Viral vectors are
constructed from one of several DNA viral expression cassettes such
as Adenovirus and Adeno-associated virus (2-3, 7, 11, 19, 21) and
Herpes Simplex Virus (5, 14-15, 18) or RNA viral expression
cassettes such as Lentivirus (6, 20, 22, 24), Sindbis Virus (9),
Murine Leukemia Virus (10, 12-13, 16) or Foamy Virus (8, 17) and
any of the biologically active RNA molecules described elsewhere in
the application and in the previous examples. For each virus, the
structural genes encoding viral coat proteins and fusogenic
proteins are subcloned into any of the pEGEN backbone plasmids for
expression from a Pol-II promoter sequence generating pVir1.
Separately, the non-structural genes encoding the polymerases and
accessory proteins are coupled with the biologically active RNA
sequence and subcloned into a second pEGEN plasmid for expression
from a Pol-II promoter sequence generating pVir2. The arrangement
of promoter sequences within pVir2 can vary for the different viral
backbones. Viral non-structural genes and templates for
biologically active RNA molecules can be expressed from either
common or independent promoter sequences endogenous to the native
virus or from within Table VIII. Plasmids pVir1 and pVir2 are
co-transfected into recipient cells to generate virus producing
cells.
[0350] Successful generation of virus producing cells can be
verified via a number of different experimental assays. Expression
of viral structural genes can be assessed using RT-PCR with primers
specific to the virus transcript and ELISAs with antibodies
specific to the viral proteins. Expression of the viral
non-structural genes can also be assessed by RT-PCR with primers
specific to the virus transcript and also with primers that bridge
the non-structural genes and the biologically active RNA. Secretion
of viral particles can be assessed by collecting the media in which
the virus producing cells are growing, isolating the protein, DNA,
or RNA from that media and then assaying for viral proteins or
nucleic acids using ELISAs, PCR, or RT-PCR. Functional viral
particles can be detected via plaque assays utilizing cell lines
carrying helper viruses.
Example 28
Administration of Viral Packaging Cells to Target Cells in
Culture
[0351] Viral packaging cells are produced from MDCK recipient cells
by transfection with pVir plasmids as described in Examples 11-14.
Virus packaging function is verified with assays described in
Example 29. In this example, the viral packaging cells produce a
replication defective virus carrying an shRNA targeting the VEGF
protein. These virus producing cells are used to knockdown the VEGF
protein in HeLa cells, providing a mechanism for distinguishing the
virus producing mouse cells from the human target cells. Depletion
of VEGF mRNA transcript in human cells and subsequent decreases in
the amount of secreted protein can be detected using species
specific primer sets in RT-PCR and species specific antibodies in
ELISAs, respectively. HeLa cells are cultured in 6 well plates in
DMEM+10% fetal bovine serum (2 mL total volume) to a density of 50%
confluence. Viral packaging cells are collected by trypsinization
and centrifugation (500.times.g for 5 minutes). The cell pellet is
resuspended in the same growth medium used for the HeLa target
cells and the cell density is measured using a hemocytometer. Viral
packaging cells are added to the HeLa target cells and the combined
culture is incubated at 37.degree. C. under 5% CO.sub.2. The
optimal ratio of viral packaging cells to target cells is
determined empirically for each system of cells and gene targets.
RNA or protein samples are collected from each cell culture 48-96
hours after addition of the viral packaging cells in order to assay
knockdown of the mRNA transcript or protein, respectively.
Example 29
Assays for Confirming the Production and Secretion of the
Recombinant Virus in Cell Culture
[0352] Cells are transfected with a pVir expression vectors or a
null vector using the methods described in Examples 11-14.
Successful generation of virus producing cells is confirmed by
assays that verify one or more of the following: (1) production of
the viral protein components, (2) production of the partial viral
genome containing the biologically active RNA template or molecule,
(3) encapsulation of the Sec-RNA into the viral particle and (4)
successful release of the viral particle from the viral production
cell. Production of the viral protein components can be verified
through RT-PCR based assays that detect the plasmid derived mRNA
transcript encoding those proteins and antibody based assays that
detect the proteins themselves. For purposes of detecting the viral
proteins, short "protein tags" which are recognized by commercially
available antibodies, can be included in the sequence of the viral
proteins. These protein tags are used to verify the function of the
viral production cell and are not necessarily included in the
functional viral particles.
[0353] To detect the plasmid derived mRNA transcript, total RNA is
prepared from pVir-transfected, null vector-transfected, and
non-transfected cells, i.e., HeLa cells or any of the other cells
described herein and otherwise known in the art, using Tri-Reagent
(Sigma-Aldrich, product #T9424) according to the manufacturer's
protocols. A cDNA library is prepared from the total RNA using a
poly-T primer and used as template for the PCR amplification.
Primers for two separate amplification reactions, each producing a
different size product, are included in the PCR reactions: (1)
Primers amplifying sequences from an internal control gene, such as
.beta.-actin or GAPDH, and (2) Primers amplifying sequences
specific to the mRNA encoding the fusion protein. Products are
resolved on 2% agarose gels run in 1.times.TAE or on 10% acrylamide
gels run in 1.times.TBE. Products are compared for the
non-transfected cells (negative control), cells transfected with a
null vector (backbone vector without the fusion protein), and the
potential viral production cells (i.e., cells transfected with a
pVir) through staining with ethidium bromide and illumination with
UV light at 302 nm. Non-transfected control cells have a single PCR
product for the internal control gene while successful BioReactors
have products for both the internal control gene and the transcript
encoding the fusion protein.
[0354] Direct detection of the viral proteins is accomplished by
collection of total protein from pVir-transfected, null
vector-transfected, and non-transfected cells, as well as the media
in which those cells are growing. Total protein is concentrated
from each sample by acetone precipitation and the concentrated
proteins are resuspended in either a native buffer for ELISA
analysis or denaturing buffer for western blot analysis. Each assay
utilizes standard methods and antibodies specific for an internal
control gene (.beta.-actin or GAPDH) and a protein tag present in
the viral protein. As discussed, protein tags are included in the
viral proteins as a convenient means for verifying function of the
viral production cell. Non-transfected and null vector-transfected
control cells have a single protein detected for the internal
control gene while successful viral production cells have both the
internal control protein and the viral proteins.
[0355] Successful production of the partial viral genome with the
inhibitory RNA template or molecule can be verified through
amplification of the DNA or RNA product. RT-PCR assays are used to
show production of the plasmid derived partial viral genome and
cellular fractionation is used to demonstrate accumulation of this
nucleic acid in the cytoplasm. The cellular fractionation is
accomplished with the PARIS RNA isolation kit (Ambion, Product
#1921) according to the manufacturer's protocol. A cDNA library is
prepared from the fractionated RNA using a random hexamer
non-specific primer and is used as template for the PCR
amplification. Primers for two separate amplification reactions,
each producing a different size product, are included in the PCR
reactions: (1) Primers amplifying sequences from an internal
control gene, such as .beta.-actin or GAPDH, and (2) Primers
amplifying sequences specific to the partial viral genome. Products
are resolved on 2% agarose gels run in 1.times.TAE or on 10%
acrylamide gels run in 1.times.TBE. Products are compared for the
null vector-transfected and non-transfected cells (negative
controls) and the potential viral production cells through staining
with ethidium bromide and illumination with UV light at 302 nm.
Null vector-transfected and non-transfected control cells have a
single PCR product for the internal control gene while successful
viral production cells have products for both the internal control
gene and the partial viral genome.
[0356] Encapsulation of the partial viral genome and inhibitory RNA
template or molecule is demonstrated through isolation of viral
particles by ultracentrifugation through CsCl gradients. Virus
particles are harvested from pVir-transfected, null
vector-transfected, and non-transfected cells and subjected to CsCl
gradient purification. Nucleic acids are prepared from the isolated
viral particles and used as template for either PCR analysis (DNA
virus backbones) or RT-PCR (RNA virus backbones) as described
above. Successful release of the viral particle is verified by
detection of the viral proteins or partial viral genome in the
extracellular matrix, or media in the case of cells in culture.
Intact viral particles can be purified and concentrated from the
media, and nucleic acids purified and used as templates for PCR or
RT-PCR analysis as described above.
Example 30
Construction of Viral Vectors Producing Recombinant Virus Carrying
Complete Bioreactor Cassettes in Cell Culture
[0357] Viral vectors are constructed from isolated plasmid
backbones, expression cassettes for the structural and
non-structural components of the virus and expression cassettes for
both the biologically active RNA and the fusion protein. PCR
amplification of expression cassettes, subcloning of expression
cassettes into plasmid backbones, amplification and isolation of
the resulting virus producing vectors and subsequent verification
of plasmid sequences are all carried out as described in Example 1.
These viral vectors utilize DNA viruses (any listed in Example 28)
such that the viral particles carry the bioreactor expression
cassettes. For each virus, the structural genes encoding viral coat
proteins and fusogenic proteins are subcloned into any of the pEGEN
backbone plasmids for expression from a Pol-II promoter sequence
generating pVir1. Separately, the non-structural genes encoding the
polymerases and accessory proteins are coupled with the expression
cassettes for the biologically active RNA(s) and the fusion protein
and subcloned into a second pEGEN plasmid for expression from a
Pol-II promoter sequence generating pVir3. Plasmids pVir1 and pVir3
are co-transfected into recipient cells to generate virus producing
cells. Cells are transfected with the pVir expression vectors or a
null vector using the methods described in Examples 11-14.
Example 31
Assays for Confirming the Production and Secretion of the
Recombinant Virus Carrying Complete Bioreactor Cassettes in Cell
Culture
[0358] Cells transfected with the pVir plasmids become viral
production cells and produce viral particles which, upon infection
of a target cell, convert that target cell into a bioreactor cell.
Successful generation of virus producing cells is confirmed by
assays that verify one or more of the following: (1) production of
the viral protein components, (2) production of the partial viral
genome containing the biologically active RNA template or molecule
as well as the template for the fusion protein, (3) encapsulation
of the biologically active RNA template and the fusion protein
template into the viral particle, (4) successful release of the
viral particle from the viral production cell and (5) successful
generation of bioreactor activity within the infected target cell.
Production of the viral protein components are verified using
assays described in Example 27. Production of the viral genomes and
bioreactor expression components are verified using assays
described in. Example 16. Encapsulation of the required nucleic
acids are verified using assays described in Example 29. Successful
release of virus particles and generation of bioreactor activity in
infected target cells are verified using assays described in
Example 16.
Example 32
Administration of the Viral Production Cells to HeLa Cells for the
Purpose of mRNA Transcript Knockdown in Cell Culture
[0359] Viral production cells, such as those produced from Examples
30-31 and confirmed using the methods described in Example 31, are
applied directly to target cells for the purpose of knocking down
the gene product targeted by the biologically active RNA molecule.
The particular pVir plasmids and recipient cells used in the
transfection are determined by the gene target of interest and the
target cell identity. In this example, the HeLa target cells are
co-cultured with MDCK viral production cells which generate viral
particles carrying expression cassettes for a bioreactor fusion
protein and an a secreted shRNA targeting VEGF (or any of the
transcripts listed in Table VII). The infected HeLa cells then
become bioreactor cells capable of producing the fusion
protein--Sec-shRNA complex and secreting that complex into the
growth media. This media can then be transferred to secondary
target cells (HeLa or other cell lines) for transfection and
subsequent VEGF knockdown. Alternatively, fusion protein--Sec-shRNA
complexes can be purified through precipitation with the 6.times.
Histidine epitope tags prior to application to the target cells. It
is possible to observe knockdown of the VEGF transcript in the
human target cells through the use of species specific primer sets
and RT-PCR. Depletion of the VEGF protein in human cells and
subsequent decreases in the amount of secreted protein can also be
detected in the media using assays with VEGF antibodies specific
for the human protein.
Example 33
Administration of Viral Packaging Cells In Vivo
[0360] Viral packaging cells are produced from NIH3T3 recipient
cells by transfection with the pVir plasmids as described in
Examples 11-14. Virus packaging function is verified with assays
described in Example 29. In this example, the NIH3T3 virus
packaging cells produce a replication defective virus carrying an
shRNA targeting the VEGF protein. The viral packaging cells are
mixed with SCCVII target cells (a mouse squamous cell carcinoma
line) and the mixture is transplanted into nude mice
(immune-compromised) by subcutaneous injection into the rear flanks
of each animal. Activity is monitored by assessment of VEGF
transcript and protein levels in tissues surrounding the
transplantation site. RNA samples are prepared from tissue
collected from the rear flanks of untreated mice, mice transplanted
with BioReactor cells secreting non-specific Sec-shRNAs and mice
transplanted with BioReactor cells secreting shRNAs targeting the
VEGF transcript using Tri-Reagent (Sigma-Aldrich, product #T9424).
Relative levels of VEGF transcript can then be assessed by RT-PCR
as described in Example 11. Viral packaging function are also
assessed in vivo by comparing tumor growth in the virus
producing/SCCVII transplants to control mice receiving SCCVII cells
alone or SCCVII cells with non-functional virus producing cells
(non-specific shRNAs or delivery compromised viruses).
Example 34
In Vivo Administration of Viral Packaging Cells to Mouse Muscle
Tissue
[0361] Viral packaging cells are produced from primary mouse
myoblast recipient cells by transfection with the pVir plasmids as
described in Examples 11-14. Virus function is verified using
assays described in Example 29. In this example, viral packaging
cells produce a replication incompetent viral particle with an
shRNAs targeting the mRNA transcript for myostatin, a negative
regulator of skeletal muscle growth. The viral packaging cells are
transplanted into the tibialis muscle of mdx mice, a model system
for Duchenne muscular dystrophy (Li S, Kimura E, Ng R, Fall B M,
Meuse L, Reyes M, Faulkner J A, Chamberlain J S., A highly
functional mini-dystrophin/GFP fusion gene for cell and gene
therapy studies of Duchenne muscular dystrophy., Hum Mol Genet.
2006 May 15; 15(10):1610-22). Virus activity is monitored by
assessment of myostatin transcript and protein levels in tissues
surrounding the transplantation site. RNA and protein samples are
prepared from tibialis muscles collected from untreated mice, mice
transplanted with viral production cells producing viral particles
with non-specific shRNAs and mice transplanted with viral packaging
cells with shRNAs targeting the myostatin transcript using
Tri-Reagent (Sigma-Aldrich, product #T9424). Relative levels of
myostatin transcript and protein can then be assessed by RT-PCR or
ELISA, respectively, as described in Example 16. Virus function is
also assessed in vivo by comparing body mass, muscle mass, muscle
size and muscle strength in the viral packaging cell transplants
relative to control mice receiving no viral packaging cells or
non-functional viral packaging cells (Bogdanovich S, Krag T O,
Barton E R, Morris L D, Whittemore L A, Ahima R S, Khurana T S.,
Functional improvement of dystrophic muscle by myostatin blockade.,
Nature. 2002 Nov. 28; 420(6914):418-21.).
Example 35
Administration of Viral Packaging Cells to Mouse Neural Tissue
[0362] Viral packaging cells are produced from mouse neural stem
cells (mNSC) by transfection with the pVir plasmid as described in
Examples 11-14. Virus function is verified with assays described in
Example 29. In this example, the mNSC viral packaging cells produce
a replication defective virus carrying an shRNA targeting the mRNA
transcript with the CAG repeat expansion of the mutant huntingtin
(htt) protein. The virus producing cells are transplanted into the
brain of mouse models for Huntington's disease to evaluate the
efficacy of virus mediated knockdown of the mRNA transcript for the
mutant form of the htt protein. RNA samples are prepared from mouse
brain tissue collected from untreated mice, mice transplanted with
viral production cells producing viral particles containing
non-specific shRNAs and mice transplanted with viral production
cells with shRNAs targeting the mutant huntingtin transcript using
Tri-Reagent (Sigma-Aldrich, product #T9424). Relative levels of
huntingtin transcript can then be assessed by RT-PCR as described
in Example 11.
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[0390] The entire disclosure of each document cited (including
patents, patent applications, journal articles, abstracts,
laboratory manuals, books, or other disclosures) is hereby
incorporated herein by reference in its entirety. Further, the
Sequence Listing submitted herewith is incorporated herein by
reference in its entirety.
[0391] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
[0392] It will be clear that the invention may be practiced
otherwise than as particularly described in the foregoing
description and examples. Numerous modifications and variations of
the invention are possible in light of the above teachings and,
therefore, are within the scope of the appended claims.
TABLE-US-00001 TABLE I Non-limiting examples of Bilogically Active
RNA Sequences SEQ ID Name Nucleotide Sequence NO 1 Mmp2
GCAAUACCUGAAUACUUUCUACUCGA 1 GUAGAAAGUAUUCAGGUAUUGC 2 VEGF
GCGGAUCAAACCUCACCAAACUCGAG 2 (shRNA) UUUGGUGAGGUUUGAUCCGCA 3 VEGF
CCAUGUACCAGCCUGGCUGAUGAGUC 3 (ribozyme) CGUGAGGACGAAAACCACUUG 4
Cav-1 GACCCACUCUUUGAAGCUGUUCUCGA 4 GAACAGCUUCAAAGAGUGGGU 5 EGFR
CUCCAUAAAUGCUACGAAUACUCGAG 5 UAUUCGUAGCAUUUAUGGAGA 6 H-Ras
CCAGGAGGAGUACAGCGCCAUCUCGA 6 GAUGGCGCUGUACUCCUCCUGG 7 Bcl-2
GGAUGACUGAGUACCUGAACCUCGAG 7 GUUCAGGUACUCAGUCAUCCA 8 Survivin
GGCUGGCUUCAUCCACUGCUUCAAGA 8 GAGCAGUGGAUGAAGCCAGCC 9 FAK
AACCACCUGGGCCAGUAUUAUCUCGA 9 GAUAAUACUGGCCCAGGUGGUU 10 STAT3
GCCGAUCUAGGCAGAUGCCACACCCAU 10 CUGCCUAGAUCGGC 11 HER3
CGCGUGUGCCAGCGAAAGUUGCGUAU 11 GGGUCACAUCGCAGGCACAUGUCAUC
UGGGCGGUCCGUUCG 12 .beta.-catenin GGACGCGUGGUACCAGGCCGAUCUAU 12
GGACGCUAUAGGCACACCGGAUACUU UAACGAUUGGCUAAGCUUCCGCGGGG AUC 13 Src
UCAGAGCGGUUACUGCUCAAUCUCGA 13 GAUUGAGCAGUAACCGCUCUGA 14 RET
GCGCGGGAAUAGUAUGGAAGGAUACG 14 UAUACCGUGCAAUCCAGGGCAACG 15
NF-.kappa.B GAUCUUGAAACUGUUUUAAGGUUGGC 15 CGAUCUU
TABLE-US-00002 TABLE II Non-limiting Examples of RNA Recognition
Sequences SEQ ID Name Nucleotide Sequence NO 1 U1 loop sequence
GGGUAUCCAUUGCACUCCGGAUGCC 16 2 Group II intron
UUUGAAGAAAAAAUAAAAGGAAUUCU 17 AUCAAUUUUUAUUUUCCAUUUAUUUA
GUUAGUUUUUCUUAAUGAAAUUGAAA UUAUUAACUAACAGAGCAAACACAAA 3 NRE stem
loop GGCCGAAAUCCCGAAGUAGGCC 18 4 S1A stem loop
GGACUGUCCACAAGACAGUCC 19 5 ARE sequence AUUUAUUUAUUUA 20 6 Box B
sequence GGCCCUGAAAAAGGGC 21 7 Rev sequence
GGUCUGGGCGCAGCGCAAGCUGCGGU 22 ACAGGCC 8 AMV sequence
GGCAUGCUCAUGCAAAACUGCAUGAA 23 UGCCCCUAAGGGAUGC
TABLE-US-00003 TABLE III Non-limiting Examples of RNA Binding
Domains SEQ ID Name Amino Acid Sequence NO 1 U1A
MAVPETRPNHTIYINNLNEKIKKDELKKS 24 LYAIFSQFGQILDILVSRSLKMRGQAFVIF
KEVSSARNALRSMQGFPFYDKPMRIQYA KTDSDIIAKMK 2 CRS1
LETHELRRLRRLARGIGRWARAKKAGVT 25 CRM1 DEVVKEVRREWASGEELAAVRIVEPLRR
SMDRAREILEIKTGGLVVWTKGDMHFV YRG 3 Nucleolin RBD
MGSHMVEGSESTTPFNLFIGNLNPNKS 26 VAELKVAISELFAKNDLAVVDVRTGTNR
KFGYVDFESAEDLEKALELTGLKVFGNE IKLEKPKGRDSKKVRAARTLLAKNLSFNI
TEDELKEVFEDALEIRLVSQDGKSKCIAYI EFKSEADAEKNLEEKQGAEIDGRSVSLYY TGEKG
4 hRBMY MVEADHPGKLTIGGLNRETNEKMLKAVF 27
GKHGPISEVLLIKDRTSKSRGFAFITFENP ADAKNAAKDMNGKSLHGKAIKVEQAKK
PSFQSGGRRRPPA 5 Tristetrapolin MSRYKTELCRTFSESGRCRYGAKCQFAH 28
TTP73 GLGELRQANRHPKYKTELCHKFYLQGRC PYGSRCHFIHNPSEDLAA 6
Bacteriophage MDAQTRRRERRAEKQAQWKAAN 29 Protein N 7 Rev
DTRQARRNRRRRWRERQRAAAAR 30 8 AMV coat SSSQKKAGGKAGKPTKRSQNYAALRK
31
TABLE-US-00004 TABLE IV Non-limiting examples of Cell Penetrating
Peptide Sequences SEQ ID Name Amino Acid Sequence NO 1 Penetratin
RQIKIWFQNRRMKWKK 32 2 Transportan GWTLNSAGYLLKINLKALAALAKKIL 33 3
MAP KLALKLALKALKALKAALKLA 34 4 TAT GRKKRRQRRRPPQ 35 5 Antp
RQIKIYFQNRRMKWKK 36 6 Rev TRQARRNRRRRWRERQR 37 7 FHV RRRNRTRRNRRRVR
38 8 TP10 AGYLLGKINLKALAALAKKIL 39 9 pVEC LLIILRRRIRKQAHAHSK 40
TABLE-US-00005 TABLE V Non-limiting examples of Non-classical
Secretory Domain Sequences SEQ ID Name Amino Acid Sequence NO 1
FGF1 MAEGEITTFAALTERFNLPLGNYKKPKLL 41 YCSNGGHFLRILPDGTVDGTRDRSDQHIQ
LQLSAESAGEVYIKGTETGQYLAMDTEG LLYGSQTPNEECLFLERLEENHYNTYTSK
KHAEKNWFVGLKKNGSCKRGPRTHYGQ KAILFLPLPVSSD 2 FGF2
MAAGSITTLPALPEDGGSGAFPPGHFKDP 42 KRLYCKNGGFFLRIHPDGRVDGVREKSD
PHIKLQLQAEERGVVSIKGVCANRYLAM KEDGRLLASRCVTDECFFFERLESNNYNT
YRSRKYTSWYVALKRTGQYKLGSKTGP GQKAILFLAMSAKS 3 Thioredoxin
MVKQIESKTAFQEALDAAGDKLVVVDFS 43 ATWCGPCKMIKPFFHSLSEKYSNVIFLEV
DVDDCQDVASECEVKCMPTFQFFKKGQ KVGEFSGANKEKLEATINELV 4 Galectin-1
MACGLVASNLNLKPGECLRVRGEVAPD 44 AKSFVLNLGKDSNNLCLHFNPRFNAHGD
ANTIVCNSKDGGAWGTEQREAVFPFQPG SVAEVCITFDQANLTVKLPDGYEFKFPNR
LNLEAINYMAADGDFKIKCVAFD 5 Galectin-3 MADNFSLHDALSGSGNPNPQGWPGAWG 45
NQPAGAGGYPGASYPGAYPGQAPPGAYP GQAPPGAYPGAPGAYPGAPAPGVYPGPP
SGPGAYPSSGQPSATGAYPATGPYGAPA GPLIVPYNLPLPGGVVPRMLITILGTVKPN
ANRIALDFQRGNDVAFHFNPRFNENNRR VIVCNTKLDNNWGREERQSVFPFESGKPF
KIQVLVEPDHFKVAVNDAHLLQYNHRV KKLNEISKLGISGDIDLTSASYTMI 6 IL-1.alpha.
MAKVPDMFEDLKNCYSENEEDSSSIDHL 46 SLNQKSFYHVSYGPLHEGCMDQSVSLSIS
ETSKTSKLTFKESMVVVATNGKVLKKRR LSLSQSITDDDLEAIANDSEEEIIKPRSAPF
SFLSNVKYNFMRIIKYEFILNDALNQSIIR ANDQYLTAAALHNLDEAVKFDMGAYKS
SKDDAKITVILRISKTQLYVTAQDEDQPV LLKEMPEIPKTITGSETNLLFFWETHGTK
NYFTSVAHPNLFIATKQDYWVCLAGGPP SITDFQILENQA 7 IL-1.beta.
MAEVPELASEMMAYYSGNEDDLFFEAD 47 GPKQMKCSFQDLDLCPLDGGIQLRISDHH
YSKGFRQAASVVVAMDKLRKMLVPCPQ TFQENDLSTFFPFIFEEEPIFFDTWDNEAY
VHDAPVRSLNCTLRDSQQKSLVMSGPYE LKALHLQGQDMEQQVVFSMSFVQGEES
NDKIPVALGLKEKNLYLSCVLKDDKPTL QLESVDPKNYPKKKMEKRFVFNKIEINN
KLEFESAQFPNWYISTSQAENMPVFLGGT KGGQDITDFTMQFVSS 8 Rhodanese
MVHQVLYRALVSTKWLAESVRAGKVGP 48 GLRVLDASWYSPGTREARKEYLERHVPG
ASFFDIEECRDKASPYEVMLPSEAGFADY VGSLGISNDTHVVVYDGDDLGSFYAPRV
WWMFRVFGHRTVSVLNGGFRNWLKEG HPVTSEPSRPEPAIFKATLNRSLLKTYEQV
LENLESKRFQLVDSRAQGRYLGTQPEPD AVGLDSGHIRGSVNMPFMNFLTEDGFEK
SPEELRAMFEAKKVDLTKPLIATCRKGVT ACHIALAAYLCGKPDVAIYDGSWFEWFH
RAPPETWVSQGKGGKA
TABLE-US-00006 TABLE VI Non-limiting examples of Fusogenic Peptide
Sequences SEQ ID Name Amino Acid Sequence NO 1 HA from
GLFGAIAGFIEGGWTGLIDG 50 influenza 2 Gp41 from HIV AVGIGALFLGFLGAAG
51 3 Melittin GIGAVLKVLTTGLPALISWIKRKRQQ 52 4 GALA
WEAALAEALAEALAEHLAEALAEALEALAA 53 5 KALA
WEAKLAKALAKALAKHLAKALAKALKACEA 54
TABLE-US-00007 TABLE VII Non-limiting examples of Targeted
Sequences and Associated Human diseases Name Disease System -
Cellular Function 1 Mmp2 Cancer Metastasis Arthritis 2 VEGF Cancer
Cell Growth/Angiogenesis Macular Degeneration 3 Cav-1 Cancer
Metastasis 4 EGFR Cancer Cell Growth 5 H-Ras Cancer 6 Bcl-2 Cancer
Cell Apoptosis/Drug Resistance 7 Survivin Cancer Cell Apoptosis 8
FAK Cancer Cell Apoptosis 9 STAT3 Cancer Cell Apoptosis 10 HER3
Cancer Cell Growth/Differentiation 11 .beta.-catenin Cancer Cell
Growth/Oncogene Activation 12 Src Cancer Cell Metastasis/Growth 13
RET Cancer Cell Growth/Survival 14 NF-.kappa.B Cancer Cell Drug
Resistance 15 Myostatin Duchennes Muscular Dystrophy 16 Huntingtin
Huntington's Disease 17 KSP Cancer Cell Division 18 MDR Cancer Cell
Drug Resistance 19 ApoB Coronary Heart Disease
TABLE-US-00008 TABLE VIII Non-limiting examples of suitable
promoters for Plasmids of the invention Name Corresponding plasmid
1 SV40 pEGEN1.1 2 Chicken .beta.-actin pEGEN2.1 3 CMV pEGEN3.1 4
Human U6 pEGEN4.1 5 Human H1 pEGEN5.1 6 Human Albumin pEGEN6.1 7
Human HIF-a pEGEN7.1 8 Human Gelsolin pEGEN8.1 9 Human CA-125
pEGEN9.1 10 Human PSA pEGEN10.1 11 Human Ubiquitin pEGEN11.1
Sequence CWU 1
1
54148RNAArtificial SequenceSynthetic 1gcaauaccug aauacuuucu
acucgaguag aaaguauuca gguauugc 48247RNAArtificial SequenceSynthetic
2gcggaucaaa ccucaccaaa cucgaguuug gugagguuug auccgca
47347RNAArtificial SequenceSynthetic 3ccauguacca gccuggcuga
ugaguccgug aggacgaaaa ccacuug 47447RNAArtificial SequenceSynthetic
4gacccacucu uugaagcugu ucucgagaac agcuucaaag agugggu
47547RNAArtificial SequenceSynthetic 5cuccauaaau gcuacgaaua
cucgaguauu cguagcauuu auggaga 47648RNAArtificial SequenceSynthetic
6ccaggaggag uacagcgcca ucucgagaug gcgcuguacu ccuccugg
48747RNAArtificial SequenceSynthetic 7ggaugacuga guaccugaac
cucgagguuc agguacucag ucaucca 47847RNAArtificial SequenceSynthetic
8ggcuggcuuc auccacugcu ucaagagagc aguggaugaa gccagcc
47948RNAArtificial SequenceSynthetic 9aaccaccugg gccaguauua
ucucgagaua auacuggccc aggugguu 481041RNAArtificial
SequenceSynthetic 10gccgaucuag gcagaugcca cacccaucug ccuagaucgg c
411167RNAArtificial SequenceSynthetic 11cgcgugugcc agcgaaaguu
gcguaugggu cacaucgcag gcacauguca ucugggcggu 60ccguucg
671281RNAArtificial SequenceSynthetic 12ggacgcgugg uaccaggccg
aucuauggac gcuauaggca caccggauac uuuaacgauu 60ggcuaagcuu ccgcggggau
c 811348RNAArtificial SequenceSynthetic 13ucagagcggu uacugcucaa
ucucgagauu gagcaguaac cgcucuga 481450RNAArtificial
SequenceSynthetic 14gcgcgggaau aguauggaag gauacguaua ccgugcaauc
cagggcaacg 501533RNAArtificial SequenceSynthetic 15gaucuugaaa
cuguuuuaag guuggccgau cuu 331625RNAArtificial SequenceSynthetic
16ggguauccau ugcacuccgg augcc 2517104RNAArtificial
SequenceSynthetic 17uuugaagaaa aaauaaaagg aauucuauca auuuuuauuu
uccauuuauu uaguuaguuu 60uucuuaauga aauugaaauu auuaacuaac agagcaaaca
caaa 1041822RNAArtificial SequenceSynthetic 18ggccgaaauc ccgaaguagg
cc 221921RNAArtificial SequenceSynthetic 19ggacugucca caagacaguc c
212013RNAArtificial SequenceSynthetic 20auuuauuuau uua
132116RNAArtificial SequenceSynthetic 21ggcccugaaa aagggc
162233RNAArtificial SequenceSynthetic 22ggucugggcg cagcgcaagc
ugcgguacag gcc 332342RNAArtificial SequenceSynthetic 23ggcaugcuca
ugcaaaacug caugaaugcc ccuaagggau gc 422498PRTArtificial
SequenceSynthetic 24Met Ala Val Pro Glu Thr Arg Pro Asn His Thr Ile
Tyr Ile Asn Asn 1 5 10 15 Leu Asn Glu Lys Ile Lys Lys Asp Glu Leu
Lys Lys Ser Leu Tyr Ala 20 25 30 Ile Phe Ser Gln Phe Gly Gln Ile
Leu Asp Ile Leu Val Ser Arg Ser 35 40 45 Leu Lys Met Arg Gly Gln
Ala Phe Val Ile Phe Lys Glu Val Ser Ser 50 55 60 Ala Arg Asn Ala
Leu Arg Ser Met Gln Gly Phe Pro Phe Tyr Asp Lys 65 70 75 80 Pro Met
Arg Ile Gln Tyr Ala Lys Thr Asp Ser Asp Ile Ile Ala Lys 85 90 95
Met Lys 2586PRTArtificial SequenceSynthetic 25Leu Glu Thr His Glu
Leu Arg Arg Leu Arg Arg Leu Ala Arg Gly Ile 1 5 10 15 Gly Arg Trp
Ala Arg Ala Lys Lys Ala Gly Val Thr Asp Glu Val Val 20 25 30 Lys
Glu Val Arg Arg Glu Trp Ala Ser Gly Glu Glu Leu Ala Ala Val 35 40
45 Arg Ile Val Glu Pro Leu Arg Arg Ser Met Asp Arg Ala Arg Glu Ile
50 55 60 Leu Glu Ile Lys Thr Gly Gly Leu Val Val Trp Thr Lys Gly
Asp Met 65 70 75 80 His Phe Val Tyr Arg Gly 85 26176PRTArtificial
SequenceSynthetic 26Met Gly Ser His Met Val Glu Gly Ser Glu Ser Thr
Thr Pro Phe Asn 1 5 10 15 Leu Phe Ile Gly Asn Leu Asn Pro Asn Lys
Ser Val Ala Glu Leu Lys 20 25 30 Val Ala Ile Ser Glu Leu Phe Ala
Lys Asn Asp Leu Ala Val Val Asp 35 40 45 Val Arg Thr Gly Thr Asn
Arg Lys Phe Gly Tyr Val Asp Phe Glu Ser 50 55 60 Ala Glu Asp Leu
Glu Lys Ala Leu Glu Leu Thr Gly Leu Lys Val Phe 65 70 75 80 Gly Asn
Glu Ile Lys Leu Glu Lys Pro Lys Gly Arg Asp Ser Lys Lys 85 90 95
Val Arg Ala Ala Arg Thr Leu Leu Ala Lys Asn Leu Ser Phe Asn Ile 100
105 110 Thr Glu Asp Glu Leu Lys Glu Val Phe Glu Asp Ala Leu Glu Ile
Arg 115 120 125 Leu Val Ser Gln Asp Gly Lys Ser Lys Cys Ile Ala Tyr
Ile Glu Phe 130 135 140 Lys Ser Glu Ala Asp Ala Glu Lys Asn Leu Glu
Glu Lys Gln Gly Ala 145 150 155 160 Glu Ile Asp Gly Arg Ser Val Ser
Leu Tyr Tyr Thr Gly Glu Lys Gly 165 170 175 2798PRTArtificial
SequenceSynthetic 27Met Val Glu Ala Asp His Pro Gly Lys Leu Thr Ile
Gly Gly Leu Asn 1 5 10 15 Arg Glu Thr Asn Glu Lys Met Leu Lys Ala
Val Phe Gly Lys His Gly 20 25 30 Pro Ile Ser Glu Val Leu Leu Ile
Lys Asp Arg Thr Ser Lys Ser Arg 35 40 45 Gly Phe Ala Phe Ile Thr
Phe Glu Asn Pro Ala Asp Ala Lys Asn Ala 50 55 60 Ala Lys Asp Met
Asn Gly Lys Ser Leu His Gly Lys Ala Ile Lys Val 65 70 75 80 Glu Gln
Ala Lys Lys Pro Ser Phe Gln Ser Gly Gly Arg Arg Arg Pro 85 90 95
Pro Ala 2874PRTArtificial SequenceSynthetic 28Met Ser Arg Tyr Lys
Thr Glu Leu Cys Arg Thr Phe Ser Glu Ser Gly 1 5 10 15 Arg Cys Arg
Tyr Gly Ala Lys Cys Gln Phe Ala His Gly Leu Gly Glu 20 25 30 Leu
Arg Gln Ala Asn Arg His Pro Lys Tyr Lys Thr Glu Leu Cys His 35 40
45 Lys Phe Tyr Leu Gln Gly Arg Cys Pro Tyr Gly Ser Arg Cys His Phe
50 55 60 Ile His Asn Pro Ser Glu Asp Leu Ala Ala 65 70
2922PRTArtificial SequenceSynthetic 29Met Asp Ala Gln Thr Arg Arg
Arg Glu Arg Arg Ala Glu Lys Gln Ala 1 5 10 15 Gln Trp Lys Ala Ala
Asn 20 3023PRTArtificial SequenceSynthetic 30Asp Thr Arg Gln Ala
Arg Arg Asn Arg Arg Arg Arg Trp Arg Glu Arg 1 5 10 15 Gln Arg Ala
Ala Ala Ala Arg 20 3126PRTArtificial SequenceSynthetic 31Ser Ser
Ser Gln Lys Lys Ala Gly Gly Lys Ala Gly Lys Pro Thr Lys 1 5 10 15
Arg Ser Gln Asn Tyr Ala Ala Leu Arg Lys 20 25 3216PRTArtificial
SequenceSynthetic 32Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met
Lys Trp Lys Lys 1 5 10 15 3326PRTArtificial SequenceSynthetic 33Gly
Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Lys Ile Asn Leu Lys 1 5 10
15 Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu 20 25 3421PRTArtificial
SequenceSynthetic 34Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys
Ala Leu Lys Ala 1 5 10 15 Ala Leu Lys Leu Ala 20 3513PRTArtificial
SequenceSynthetic 35Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro
Gln 1 5 10 3616PRTArtificial SequenceSynthetic 36Arg Gln Ile Lys
Ile Tyr Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15
3717PRTArtificial SequenceSynthetic 37Thr Arg Gln Ala Arg Arg Asn
Arg Arg Arg Arg Trp Arg Glu Arg Gln 1 5 10 15 Arg 3814PRTArtificial
SequenceSynthetic 38Arg Arg Arg Asn Arg Thr Arg Arg Asn Arg Arg Arg
Val Arg 1 5 10 3921PRTArtificial SequenceSynthetic 39Ala Gly Tyr
Leu Leu Gly Lys Ile Asn Leu Lys Ala Leu Ala Ala Leu 1 5 10 15 Ala
Lys Lys Ile Leu 20 4018PRTArtificial SequenceSynthetic 40Leu Leu
Ile Ile Leu Arg Arg Arg Ile Arg Lys Gln Ala His Ala His 1 5 10 15
Ser Lys 41155PRTArtificial SequenceSynthetic 41Met Ala Glu Gly Glu
Ile Thr Thr Phe Ala Ala Leu Thr Glu Arg Phe 1 5 10 15 Asn Leu Pro
Leu Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser 20 25 30 Asn
Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly 35 40
45 Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu
50 55 60 Ser Ala Gly Glu Val Tyr Ile Lys Gly Thr Glu Thr Gly Gln
Tyr Leu 65 70 75 80 Ala Met Asp Thr Glu Gly Leu Leu Tyr Gly Ser Gln
Thr Pro Asn Glu 85 90 95 Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu
Asn His Tyr Asn Thr Tyr 100 105 110 Thr Ser Lys Lys His Ala Glu Lys
Asn Trp Phe Val Gly Leu Lys Lys 115 120 125 Asn Gly Ser Cys Lys Arg
Gly Pro Arg Thr His Tyr Gly Gln Lys Ala 130 135 140 Ile Leu Phe Leu
Pro Leu Pro Val Ser Ser Asp 145 150 155 42155PRTArtificial
SequenceSynthetic 42Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu
Pro Glu Asp Gly 1 5 10 15 Gly Ser Gly Ala Phe Pro Pro Gly His Phe
Lys Asp Pro Lys Arg Leu 20 25 30 Tyr Cys Lys Asn Gly Gly Phe Phe
Leu Arg Ile His Pro Asp Gly Arg 35 40 45 Val Asp Gly Val Arg Glu
Lys Ser Asp Pro His Ile Lys Leu Gln Leu 50 55 60 Gln Ala Glu Glu
Arg Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn 65 70 75 80 Arg Tyr
Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Arg Cys 85 90 95
Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr 100
105 110 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu
Lys 115 120 125 Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro
Gly Gln Lys 130 135 140 Ala Ile Leu Phe Leu Ala Met Ser Ala Lys Ser
145 150 155 43105PRTArtificial SequenceSynthetic 43Met Val Lys Gln
Ile Glu Ser Lys Thr Ala Phe Gln Glu Ala Leu Asp 1 5 10 15 Ala Ala
Gly Asp Lys Leu Val Val Val Asp Phe Ser Ala Thr Trp Cys 20 25 30
Gly Pro Cys Lys Met Ile Lys Pro Phe Phe His Ser Leu Ser Glu Lys 35
40 45 Tyr Ser Asn Val Ile Phe Leu Glu Val Asp Val Asp Asp Cys Gln
Asp 50 55 60 Val Ala Ser Glu Cys Glu Val Lys Cys Met Pro Thr Phe
Gln Phe Phe 65 70 75 80 Lys Lys Gly Gln Lys Val Gly Glu Phe Ser Gly
Ala Asn Lys Glu Lys 85 90 95 Leu Glu Ala Thr Ile Asn Glu Leu Val
100 105 44135PRTArtificial SequenceSynthetic 44Met Ala Cys Gly Leu
Val Ala Ser Asn Leu Asn Leu Lys Pro Gly Glu 1 5 10 15 Cys Leu Arg
Val Arg Gly Glu Val Ala Pro Asp Ala Lys Ser Phe Val 20 25 30 Leu
Asn Leu Gly Lys Asp Ser Asn Asn Leu Cys Leu His Phe Asn Pro 35 40
45 Arg Phe Asn Ala His Gly Asp Ala Asn Thr Ile Val Cys Asn Ser Lys
50 55 60 Asp Gly Gly Ala Trp Gly Thr Glu Gln Arg Glu Ala Val Phe
Pro Phe 65 70 75 80 Gln Pro Gly Ser Val Ala Glu Val Cys Ile Thr Phe
Asp Gln Ala Asn 85 90 95 Leu Thr Val Lys Leu Pro Asp Gly Tyr Glu
Phe Lys Phe Pro Asn Arg 100 105 110 Leu Asn Leu Glu Ala Ile Asn Tyr
Met Ala Ala Asp Gly Asp Phe Lys 115 120 125 Ile Lys Cys Val Ala Phe
Asp 130 135 45250PRTArtificial SequenceSynthetic 45Met Ala Asp Asn
Phe Ser Leu His Asp Ala Leu Ser Gly Ser Gly Asn 1 5 10 15 Pro Asn
Pro Gln Gly Trp Pro Gly Ala Trp Gly Asn Gln Pro Ala Gly 20 25 30
Ala Gly Gly Tyr Pro Gly Ala Ser Tyr Pro Gly Ala Tyr Pro Gly Gln 35
40 45 Ala Pro Pro Gly Ala Tyr Pro Gly Gln Ala Pro Pro Gly Ala Tyr
Pro 50 55 60 Gly Ala Pro Gly Ala Tyr Pro Gly Ala Pro Ala Pro Gly
Val Tyr Pro 65 70 75 80 Gly Pro Pro Ser Gly Pro Gly Ala Tyr Pro Ser
Ser Gly Gln Pro Ser 85 90 95 Ala Thr Gly Ala Tyr Pro Ala Thr Gly
Pro Tyr Gly Ala Pro Ala Gly 100 105 110 Pro Leu Ile Val Pro Tyr Asn
Leu Pro Leu Pro Gly Gly Val Val Pro 115 120 125 Arg Met Leu Ile Thr
Ile Leu Gly Thr Val Lys Pro Asn Ala Asn Arg 130 135 140 Ile Ala Leu
Asp Phe Gln Arg Gly Asn Asp Val Ala Phe His Phe Asn 145 150 155 160
Pro Arg Phe Asn Glu Asn Asn Arg Arg Val Ile Val Cys Asn Thr Lys 165
170 175 Leu Asp Asn Asn Trp Gly Arg Glu Glu Arg Gln Ser Val Phe Pro
Phe 180 185 190 Glu Ser Gly Lys Pro Phe Lys Ile Gln Val Leu Val Glu
Pro Asp His 195 200 205 Phe Lys Val Ala Val Asn Asp Ala His Leu Leu
Gln Tyr Asn His Arg 210 215 220 Val Lys Lys Leu Asn Glu Ile Ser Lys
Leu Gly Ile Ser Gly Asp Ile 225 230 235 240 Asp Leu Thr Ser Ala Ser
Tyr Thr Met Ile 245 250 46271PRTArtificial SequenceSynthetic 46Met
Ala Lys Val Pro Asp Met Phe Glu Asp Leu Lys Asn Cys Tyr Ser 1 5 10
15 Glu Asn Glu Glu Asp Ser Ser Ser Ile Asp His Leu Ser Leu Asn Gln
20 25 30 Lys Ser Phe Tyr His Val Ser Tyr Gly Pro Leu His Glu Gly
Cys Met 35 40 45 Asp Gln Ser Val Ser Leu Ser Ile Ser Glu Thr Ser
Lys Thr Ser Lys 50 55 60 Leu Thr Phe Lys Glu Ser Met Val Val Val
Ala Thr Asn Gly Lys Val 65 70 75 80 Leu Lys Lys Arg Arg Leu Ser Leu
Ser Gln Ser Ile Thr Asp Asp Asp 85 90 95 Leu Glu Ala Ile Ala Asn
Asp Ser Glu Glu Glu Ile Ile Lys Pro Arg 100 105 110 Ser Ala Pro Phe
Ser Phe Leu Ser Asn Val Lys Tyr Asn Phe Met Arg 115 120 125 Ile Ile
Lys Tyr Glu Phe Ile Leu Asn Asp Ala Leu Asn Gln Ser Ile 130 135 140
Ile Arg Ala Asn Asp Gln Tyr Leu Thr Ala Ala Ala Leu His Asn Leu 145
150 155 160 Asp Glu Ala Val Lys Phe Asp Met Gly Ala Tyr Lys Ser Ser
Lys Asp 165 170 175 Asp Ala Lys Ile Thr Val Ile Leu Arg Ile Ser Lys
Thr Gln Leu Tyr 180 185 190 Val Thr Ala Gln Asp Glu Asp Gln Pro Val
Leu Leu Lys Glu Met Pro 195 200 205 Glu Ile Pro Lys Thr Ile Thr Gly
Ser Glu Thr Asn Leu Leu Phe Phe 210 215 220 Trp Glu Thr His Gly Thr
Lys Asn Tyr Phe Thr Ser Val Ala His Pro 225 230 235 240 Asn Leu Phe
Ile Ala Thr Lys Gln Asp Tyr Trp Val Cys Leu Ala Gly 245 250 255 Gly
Pro Pro
Ser Ile Thr Asp Phe Gln Ile Leu Glu Asn Gln Ala 260 265 270
47269PRTArtificial SequenceSynthetic 47Met Ala Glu Val Pro Glu Leu
Ala Ser Glu Met Met Ala Tyr Tyr Ser 1 5 10 15 Gly Asn Glu Asp Asp
Leu Phe Phe Glu Ala Asp Gly Pro Lys Gln Met 20 25 30 Lys Cys Ser
Phe Gln Asp Leu Asp Leu Cys Pro Leu Asp Gly Gly Ile 35 40 45 Gln
Leu Arg Ile Ser Asp His His Tyr Ser Lys Gly Phe Arg Gln Ala 50 55
60 Ala Ser Val Val Val Ala Met Asp Lys Leu Arg Lys Met Leu Val Pro
65 70 75 80 Cys Pro Gln Thr Phe Gln Glu Asn Asp Leu Ser Thr Phe Phe
Pro Phe 85 90 95 Ile Phe Glu Glu Glu Pro Ile Phe Phe Asp Thr Trp
Asp Asn Glu Ala 100 105 110 Tyr Val His Asp Ala Pro Val Arg Ser Leu
Asn Cys Thr Leu Arg Asp 115 120 125 Ser Gln Gln Lys Ser Leu Val Met
Ser Gly Pro Tyr Glu Leu Lys Ala 130 135 140 Leu His Leu Gln Gly Gln
Asp Met Glu Gln Gln Val Val Phe Ser Met 145 150 155 160 Ser Phe Val
Gln Gly Glu Glu Ser Asn Asp Lys Ile Pro Val Ala Leu 165 170 175 Gly
Leu Lys Glu Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp 180 185
190 Lys Pro Thr Leu Gln Leu Glu Ser Val Asp Pro Lys Asn Tyr Pro Lys
195 200 205 Lys Lys Met Glu Lys Arg Phe Val Phe Asn Lys Ile Glu Ile
Asn Asn 210 215 220 Lys Leu Glu Phe Glu Ser Ala Gln Phe Pro Asn Trp
Tyr Ile Ser Thr 225 230 235 240 Ser Gln Ala Glu Asn Met Pro Val Phe
Leu Gly Gly Thr Lys Gly Gly 245 250 255 Gln Asp Ile Thr Asp Phe Thr
Met Gln Phe Val Ser Ser 260 265 48297PRTArtificial
SequenceSynthetic 48Met Val His Gln Val Leu Tyr Arg Ala Leu Val Ser
Thr Lys Trp Leu 1 5 10 15 Ala Glu Ser Val Arg Ala Gly Lys Val Gly
Pro Gly Leu Arg Val Leu 20 25 30 Asp Ala Ser Trp Tyr Ser Pro Gly
Thr Arg Glu Ala Arg Lys Glu Tyr 35 40 45 Leu Glu Arg His Val Pro
Gly Ala Ser Phe Phe Asp Ile Glu Glu Cys 50 55 60 Arg Asp Lys Ala
Ser Pro Tyr Glu Val Met Leu Pro Ser Glu Ala Gly 65 70 75 80 Phe Ala
Asp Tyr Val Gly Ser Leu Gly Ile Ser Asn Asp Thr His Val 85 90 95
Val Val Tyr Asp Gly Asp Asp Leu Gly Ser Phe Tyr Ala Pro Arg Val 100
105 110 Trp Trp Met Phe Arg Val Phe Gly His Arg Thr Val Ser Val Leu
Asn 115 120 125 Gly Gly Phe Arg Asn Trp Leu Lys Glu Gly His Pro Val
Thr Ser Glu 130 135 140 Pro Ser Arg Pro Glu Pro Ala Ile Phe Lys Ala
Thr Leu Asn Arg Ser 145 150 155 160 Leu Leu Lys Thr Tyr Glu Gln Val
Leu Glu Asn Leu Glu Ser Lys Arg 165 170 175 Phe Gln Leu Val Asp Ser
Arg Ala Gln Gly Arg Tyr Leu Gly Thr Gln 180 185 190 Pro Glu Pro Asp
Ala Val Gly Leu Asp Ser Gly His Ile Arg Gly Ser 195 200 205 Val Asn
Met Pro Phe Met Asn Phe Leu Thr Glu Asp Gly Phe Glu Lys 210 215 220
Ser Pro Glu Glu Leu Arg Ala Met Phe Glu Ala Lys Lys Val Asp Leu 225
230 235 240 Thr Lys Pro Leu Ile Ala Thr Cys Arg Lys Gly Val Thr Ala
Cys His 245 250 255 Ile Ala Leu Ala Ala Tyr Leu Cys Gly Lys Pro Asp
Val Ala Ile Tyr 260 265 270 Asp Gly Ser Trp Phe Glu Trp Phe His Arg
Ala Pro Pro Glu Thr Trp 275 280 285 Val Ser Gln Gly Lys Gly Gly Lys
Ala 290 295 4949DNAArtificial SequenceSynthetic 49ttggcttcct
cctggttatg ttcaagagac ataaccagga ggaagccaa 495020PRTArtificial
SequenceSynthetic 50Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly
Gly Trp Thr Gly 1 5 10 15 Leu Ile Asp Gly 20 5116PRTArtificial
SequenceSynthetic 51Ala Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu
Gly Ala Ala Gly 1 5 10 15 5226PRTArtificial SequenceSynthetic 52Gly
Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu 1 5 10
15 Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln 20 25 5330PRTArtificial
SequenceSynthetic 53Trp Glu Ala Ala Leu Ala Glu Ala Leu Ala Glu Ala
Leu Ala Glu His 1 5 10 15 Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu
Ala Leu Ala Ala 20 25 30 5430PRTArtificial SequenceSynthetic 54Trp
Glu Ala Lys Leu Ala Lys Ala Leu Ala Lys Ala Leu Ala Lys His 1 5 10
15 Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Ala Cys Glu Ala 20 25
30
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