Link: Pharm/Biotech Resources
Title: Nucleic acids comprising a post-transcriptional
regulatory element (PRE) and their uses
United States Patent: 6,919,442
Issued: July 19, 2005
Inventors: Pavlakis; George N. (Rockville, MD); Nappi; Filomena (Rome, IT)
Assignee: The United States of America as represented by the Department of Health and Human Services (Washington, DC)
Appl. No.: 673716
Filed: May 18, 1999
PCT Filed: May 18, 1999
PCT NO: PCT/US99/11082
371 Date: February 26, 2001
102(e) Date: February 26, 2001
PCT PUB.NO.: WO99/61596
PCT PUB. Date: December 2, 1999
Abstract
The invention provides a novel post-transcriptional regulatory element that can function as an RNA nucleo-cytoplasmic transport element. The invention also provides for an attenuated HIV-1 hybrid virus for use as a vaccine and a kit incorporating the hybrid virus. The kit also includes instructional material teaching the use of the vaccine, where the instructional material indicates that the vaccine is used for the prophylaxis or amelioration of HIV-1 infection in a mammal; that the vaccine is to be administered to a mammal in a therapeutically effective amount sufficient to express a viral protein; where the vaccine will not cause clinically significant CD4+ cell depletion; and, the expression of the viral protein elicits an immune response to the attenuated HIV-1 virus. The invention further provides for a method for screening for post-transcriptional RNA nucleo-cytoplasmic transport element (NCTE) binding proteins.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a novel family of post-transcriptional
regulatory elements, or PRE. Specifically, the PREs of the invention can
function as RNA nucleo-cytoplasmic transport elements (NCTE). The exemplary
PRE of the invention (SEQ ID NO:1) was initially derived from a mouse
genomic nucleic acid. Sequence comparison analysis shows that PRE sequences
are highly homologous to intracisternal A particle (IAP) sequences.
In retroviruses, including HIV type 1 (HIV-1), simian retrovirus type 1
(SRV-1), SRV-2, and Mason-Pfizer monkey virus (MPMV) (the later three are
type D simian retroviruses), the rate of viral growth can be controlled by
changing the rate of expression of their RNA message. This strategy is a
"post-transcriptional" means to regulate gene expression and viral growth.
Specifically, these retroviruses need a special sequence on their RNA to
effect nuclear transport of unspliced mRNA encoding structural proteins.
This sequence, called a "post-transcriptional regulatory element" (PRE),
typically acts by internally base-pairing, allowing the RNA molecule to fold
into a unique, secondary structure (a "cis-acting element"). The folding
patterns are highly structured and are commonly stem-loop, or hairpin
structures. A soluble protein binds specifically to this RNA structure to
aid in the transport of the message from the nucleus to the cytoplasm (in
addition to other functions, including aiding in the splicing of the
transcript).
Some retroviruses, such as Simian type D retroviruses, including SRV-1, do
not encode their own trans-acting, NCTE-binding proteins and instead utilize
cellular NCTE binding proteins. Other retroviruses, such as HIV-1, utilize a
retrovirally-encoded NCTE RNA binding protein, called "Rev." HIV-1 regulates
the expression of its structural proteins encoded by the gag/pol- and env-encoding
transcript using this NCTE system. HIV-1's NCTE binding protein "Rev"
interacts with a specific NCTE sequence, designated the "Rev-responsive
element," or "RRE," contained in its gag/pol and env encoding transcript.
HIV-1's RRE does not bind cellular NCTE-binding proteins. Rev interacts
directly with RRE as part of the RNA export machinery which transports RRE-containing
transcripts to the cytoplasm from the nucleus. As a result, Rev and RRE are
needed to produce infectious virus.
HIV-1 lacking a functional Rev/RRE control system, while uninfectious, can
be reconstituted with exogenous control elements. For example, when simian
retroviral CTE (e.g., CTE from SRV-1, SRV-2, MPMV) is used to reconstitute
HIV-1's NCTE, the hybrid produces transcripts and infectious virions (Bray
(1994) Proc. Nail. Acad. Sci. USA 91:1256-1260; Tabernero (1996)
J. Virol. 70:5998-6011; Zolotukhin (1994) J. Virol.
68:7944-7952). Significantly, these hybrids produce lower levels of
transcript and productive virion than seen with wild-type HIV-1.
It was surprisingly found that the novel PRE of the invention can
functionally replace the NCTE of HIV-1, or "RRE." It was discovered that
when PRE is used in place of RRE to construct an HIV-1 hybrid clone, a
slower growing virus results. Significantly, while capable of producing
infectious virions in viva, this HIV-1 hybrid (a PRE-containing RRE-negative
recombinant virus) has lower replicative activity than wild-type virus,
resulting in an attenuated HIV-1 strain. The level of attenuation can be
quantitated using in vitro or in vivo assays, such as determining the amount
of HIV-1 p24gag synthesized by hybrid viruses. For example,
PRE-containing HIV-1 hybrids can be used to infect tissue culture cells or
activated human peripheral blood mononuclear cells (huPBMCs) in vitro. When
the PRE-containing hybrid HIV-1 virus infects activated huPBMCs, the level
of expression of HIV-1 p24gag is between about 50 fold and about
200 fold less than levels of p24gag expression when
HIV-1 wild type virus, utilizing wild-type NCTE (i.e., RRE), infects
activated huPBMCs.
The efficacy of the PRE-recombinant hybrid as an attenuating agent in HIV-1
infection and AIDS pathogenesis can be demonstrated using functionally
analogous NCTEs, such as the SRV-1 CTE. Recombinant, hybrid HIV-1 in which
NCTE from SRV-1 (termed "CTF") functionally replaces wild-type HIV-1 NCTE ("RRE")
are functionally analogous to hybrid HIV-1 clones whose RRE is replaced by
the PRE of the invention. PRE-hybrid and SRV-1 CTE-hybrid HIV-1 clones have
approximately the same rate of propagation and infectivity, as demonstrated
by the in vitro experiments discussed in Example 1. Thus, CTE attenuated
HIV-1 clones can be used to demonstrate the replicative, yet non-cytopathic,
effect of CTE-attenuated HIV-1 in the SCID-hu mouse model, as discussed in
Example 3.
CTE(+)/RRE(-;) HIV-1 clones were used to infect Thy/Liv implants, which are
human thymus and liver cells transplanted in SCID-hu mice (see, e.g.,
Kollmann (1995) J. Immunol. 154:907-921). Significantly, these
viruses propagated slower than both wild-type and Nef-negative HIV-1 clones.
This demonstrates that they have lower replicative capacity in human
lymphocytes. Furthermore, the CTE(+)/RRE(-;) attenuated HIV-1 clones were
not lympliocytopathic, no depletion of CD4+-bearing cells was
observed. This demonstrates that slow growing HIV-1 hybrid clones utilizing
exogenous NCTEs, as CTE (of SRV-1) or PRE, have an attenuated phenotype for
cytotoxicity.
Analogously, when the PRE-attenuated HIV-1 of the invention infect activated
human lymphocytes in vivo, they will also produce low levels of infectious
virions without any lymphocytotoxic effects, i.e., levels of CD4+
T cells will not decline. Importantly, this CTEIAP-attenuated
virus will elicit an immune response in the infected, yet asymptomatic,
individual.
The finding that an IAP element can be utilized in the attenuation of a
retrovirus whose productive infection does not lead to loss of CD4+
cells is especially unexpected in view of past findings that a human IAP has
been found to be associated with CD4+ T-cell immunodeficiency and
dysfunction. The presence of IAP sequence has also been associated with the
occurrence of carcinogenesis.
This invention also provides for a vaccine in the form of a pharmacological
compositions and a kit. The pharmacological compositions can comprise a
pharmaceutically acceptable carrier and the attenuated virus of the
invention. The kit can comprise a container containing a vaccine
formulation.
I. Characterization and Isolation of Nucleic Acids Encoding PRE
This invention provides for the characterization, cloning and expression of
a novel NCTE, the PRE of the invention. Initially derived from murine
genomic sequence, it is homologous to intracistemal A particles (IAPs). The
invention also provides for novel means of expressing the PRE of the
invention in vitro and in vivo. In a further embodiment, these expression
systems provide a means to screen for novel NCTEs.
The invention can be practiced in conjunction with any method or protocol
known in the art, which are well described in the scientific and patent
literature. Therefore, only a few general techniques will be described prior
to discussing specific methodologies and examples relative to the novel
reagents and methods of the invention.
A. General Techniques
Methods of isolating total DNA or RNA encoding the nucleic acids of the
invention are well known to those of skill in the art. Techniques for
isolation, purification and manipulation of nucleic acids, genes and CTEIAP
sequences, such as generating libraries, subcloning into expression vectors,
labeling probes, DNA hybridization, and the like are described, e.g., in
Sambrook, M
The nucleic acids of this invention, whether RNA, mRNA, DNA, cDNA, genomic
DNA, or a hybrid of the genetic recombinations, may be isolated from a
variety of sources or may be synthesized in vitro. Nucleic acids of the
invention can be expressed in transgenic animals, transformed cells, in a
transformed cell lysate, or in a partially purified or a substantially pure
form. Sequencing methods typically use dideoxy sequencing (Sequenase, U.S.
Biochemical), however, other kits and methods are available and well known
to those of skill in the art.
Nucleic acids and proteins are detected and quantified in accordance with
the teachings and methods of the invention. described herein by any of a
number of general means well known to those of skill in the art. These
include, for example, analytical biochemical methods such as
spectrophotometry, radiography, electrophoresis, capillary electrophoresis,
high performance liquid chromatography (HPLC), thin layer chromatography
(TLC), and hyperdiffusion chromatography, various immunological methods,
such as fluid or gel precipitin reactions, immunodiffusion (single or
double), immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked
immunosorbent assays (ELISAs), immuno-fluorescent assays, and the like,
Southern analysis, Northern analysis, Dot-blot analysis, gel
electrophoresis, RT-PCR, quantitative PCR, other nucleic acid or target or
signal amplification methods, radiolabeling, scintillation counting, and
affinity chromatography, to name only a few.
B. Identification, Synthesis and Purification of PRE Nucleic Acids
The invention provides means to identify, synthesize and purify PRE of the
invention and its alleles, isoforms and polymorphisms.
1. Preparation and Screening of DNA Libraries
There are numerous methods for isolating the DNA sequences encoding the PRE
of the invention. For example, DNA can be isolated from a genomic or cDNA
library using labeled oligonucleotide probes having sequences complementary
to the sequences or subsequences disclosed herein, such as SEQ ID NO:1. Such
probes can be used directly in hybridization assays to isolate DNA encoding
PRE isoforms and polymorphisms. Alternatively, probes can be designed for
use in amplification techniques, such as, e.g., PCR. PRE nucleic acid can be
identified and produced using such amplification methods, as described
herein.
To prepare a cDNA library, mRNA is isolated, reverse transcribed from the
mRNA according to procedures well known in the art The cDNA can be inserted
into any expression cassette or vector. The cassettes or vectors are
transfected into a recombinant host for propagation, screening and cloning.
Methods for making and screening cDNA libraries are well known. See, e.g.,
Gubler (1983) Gene 25:263-269, Sambrook, Ausubel.
To make a genomic library, total DNA is extracted and purified by well-known
methods (see, e.g., Sambrook). DNA of appropriate size is produced by known
methods, such as mechanical shearing or enzymatic digestion, to yield DNA
fragments, e.g., of about 12 to 20 kb. The fragments are then separated, as
for example, by gradient centrifugation, or gel electrophoresis, from
undesired sizes. Selected fragments can be inserted in bacteriophage,
expression cassettes, or other vectors. Recombinant phage can be analyzed by
plaque hybridization described, e.g., in Benton (1977) Science 196:
180; Chen (1997) Methods Mol Biol 62:199-206. Colony hybridization is
generally described in, e.g., Grunstein (1975) Proc. Natl. Acad. Sci. USA
72:3961-3965; Yoshioka (1997) J. Immunol Methods 201:145-155;
Palkova (1996) Biotechniques 21:982.
DNA encoding an PRE can be identified in either cDNA or genomic libraries by
hybridization with nucleic acid probes of the invention. For example, a
probe containing 10 to 20 to 50 or more contiguous nucleotides of SEQ ID
NO:1 is used in Southern blots to identify a PRE of the invention. Once
identified, these DNA regions are isolated by standard methods familiar to
those of skill in the art. Alternatively, RNA may be identified by
hybridization to nucleic acid probes in Northern blots or other formats;
see, e.g., Sambrook, Ausubel, for general procedures.
Oligonucleotides for use as, e.g., probes, templates for further
amplification, and the like, can be chemically synthesized, as described
below. Synthetic nucleic acids, including oligonucleotide probes and
primers, PRE sequences, and the like, can be prepared by a variety of
solution or solid phase methods. Detailed descriptions of the procedures for
solid phase synthesis of nucleic acids by phosphite-triester,
phosphotriester, and H-phosphonate chemistries are widely available. For
example, the solid phase pliosphoramidite triester method of Beaucage and
Carruthers using an automated synthesizer is described in Itakura, U.S. Pat.
No. 4,401,796; Carruthers, U.S. Pat. Nos. 4,458,066 and 4,500,707;
Carruthers (1982) Genetic Engineering 4:1-17. See also Needham-VanDevanter
(1984) Nucleic Acids Res. 12:6159-6168; Beigelman (1995) Nucleic
Acids Res 23: 3989-3994; O
2. Amplification of Nucleic Acids
The present invention provides oligonucleotide primers and probes that can
hybridize specifically to and amplify nucleic acids having PRE sequences.
Such reagents can be used to identify further PRE species, such as
polymorphisms alleles and other variations. For illustrative purposes,
exemplary PCR primers and amplification methods are described herein.
For amplification of PRE, nucleic acid conserved amongst different PRE
species are preferred reagents for use as hybridization and amplification
probes to identify and isolate additional species from various organisms.
Oligonucleotides can be used to identify and detect additional PRE species
using a variety of hybridization techniques and conditions. Suitable
amplification methods include, e.g., polymerase chain reaction, PCR (PCR P
The invention provides for amplification and manipulation or detection of
the products from each of the above methods to prepare DNA encoding PRE
nucleic acid. In PCR techniques, oligonucleotide primers complementary to
the two borders of the DNA region to be amplified are synthesized and used
(see, Innis). PCR can be used in a variety of protocols to amplify,
identify, quantify, isolate and manipulate nucleic acids encoding PRE. In
these protocols, primers and probes for amplification and hybridization are
generated that comprise all or any portion of the DNA sequences listed
herein An illustrative primer pair that can amplify the PRE of the invention
under appropriate conditions includes an oligonucleotide incorporating about
the first twenty or thirty nucleic acids of the exemplary PRE of the
invention, i.e., 5′-GTGGGGTGCG AGGCTAAGCA CTGCACAGAG-3′, the 5′ thirty
nucleotides of SEQ ID NO:1; and, an oligonucleotide complementary to the 3′
twenty to thirty nucleic acids, i.e., 5′-AAGCAAGCCT CATGGGTGAA GGTAGAGGAC-3′
(SEQ ID NO:2).
PCR-amplified sequences can also be labeled and used as detectable
oligonucleotide probes, but such nucleic acid probes can be generated using
any synthetic or other technique well known in the art, as described above.
The labeled amplified DNA or other oligonucleotide or nucleic acid of the
invention can be used as probes to further identify and isolate PRE species
from various cDNA or genomic libraries.
Another useful means of obtaining nucleic acids of the invention, such as
large genomic clones, is to screen YAC, BAC or P1 genomic libraries. BACs,
bacterial artificial chromosomes, are vectors that can contain 120+Kb
inserts. BACs are based on the E. coli F factor plasmid system and
simple to manipulate and purify in microgram quantities. Because BAC
plasrnids are kept at one to two copies per cell, the problems of
rearrangement observed with YACs, which can also be employed in the present
methods, are eliminated. BAC vectors can include marker genes for luciferase
and green fluorescent protein (GFP). (Baker (1997) Nucleic Acids Res
25:1950-1956). Yeast artificial chromosomes, or YACS, can also be used for
contain inserts ranging in size from 80 to 700 kb, see, e.g., Tucker (1997)
Gene 199:25-30; Adam (1997) Plant J. 11:1349-1358. P1 is a
bacteriophage that infects E. coli that can contain 75-100 Kb DNA
inserts (Mejia (1997) Genome Res 7:179-186; Ioannou (1994) Nat
Genet 6:84-89), and are screened in much the same way as lambda
libraries.
3. Cloning PRE-Encoding Inserts
The invention also provides PRE-encoding expression cassettes and vectors to
produce large quantities of full or partial length PRE nucleic acid. The
expression vectors and cassettes include, e.g., those used in bacterial,
yeast, plant, insect, in vitro, or mammalian systems. For example,
generation of PRE in this manner is useful for assaying for PRE activity
modulators, analysis of the activity of newly isolated species of PRE,
identifying and isolating compounds which specifically associate with PRE,
such as binding proteins, or analysis of the activity of PRE which has been
site-specifically mutated. The nucleic acids of the invention can also be
used as immunogens, as a few examples, see, e.g., Radic (1994) Annu. Rev.
Immunol. 12:487-520; Cabral (1997) Curr. Opin. Rheumatol.
9:387-392; Pisetsky (1997) Methods 11:55-61; Marion (1997) Methods
11:3-11, for general discussion on anti-DNA antibodies; for discussion
on generation of anti-RNA antibodies using combinatorial phage display
libraries see Marchbank (1995) Nucleic Acids Symp. Ser. 33:120-122.
There are several well-known methods of introducing nucleic acids into
bacterial and other cells, a process often called "transforming," any of
which may be used in the methods of the present invention (see, e.g.,
Sambrook). Techniques for transforming a wide variety of animal and plant
cells are well known and described in the technical and scientific
literature. See, e.g., Weising (1988) Ann. Rev. Genet. 22:421-477, for plant
cells and Sambrook for animal and bacterial cells.
4. Sequencing of PRE-Encoding Nucleic Acid
Sequencing of newly isolated DNA will identify and characterize PRE-encoding
nucleic acid of the invention. Sequencing of isolated PRE-encoding nucleic
acid can be used to identify, in addition to functional criteria, new
PRE-encoding species or allelic variations. Secondary structures can be
identified. For example, in terms of primary sequence criteria, a nucleic
acid is a PRE specie within the scope of the claimed invention if its
sequence has least 80% nucleic acid sequence identity to SEQ ID NO:1.
PRE-encoding nucleic acid sequences can be sequenced as inserts in vectors,
as inserts released and isolated from the vectors or in any of a variety of
other forms (i.e., as amplification products). PRE-encoding inserts can be
released from the vectors by restriction enzymes or amplified by PCR or
transcribed by a polymerase. For sequencing of the inserts to identify full
length PRE coding sequences, primers based on the N- or C-terminus, or based
on insertion points in the original phage or other vector, can be used.
Additional primers can be synthesized to provide overlapping sequences.
A variety of nucleic acid sequencing techniques are well known and described
in the scientific and patent literature, e.g., see Rosenthal (1987) supra;
Arlinghaus (1997) Anal. Chem. 69:3747-3753, for use of biosensor
chips for sequencing; Pastinen (1996) Clin. Chem. 42:1391-1397; Nyren
(1993) Anal Biochem. 208:171-175.
5. Nucleic Acid Hybridization Techniques
The hybridization techniques disclosed herein can be utilized to identify,
isolate and characterize amplicon-encoding nucleic acid of the invention,
including different isoforms, alleles and polymorphisms of such sequences. A
variety of methods for specific DNA and RNA measurement using nucleic acid
hybridization techniques are known to those of skill in the art. See. e.g.,
N
One method for evaluating the presence or absence of DNA encoding PRE of the
invention in a sample involves a Southern transfer. Briefly, the digested
bacterial genomic DNA is run on agarose slab gels in buffer and transferred
to membranes. Hybridization is carried out using nucleic acid probes. The
nucleic acid probes can be designed based on conserved nucleic acid
sequences. Preferably nucleic acid probes are 20 bases or longer in length
(see, e.g., Sambrook for methods of selecting nucleic acid probe sequences
for use in nucleic acid hybridization). Visualization of the hybridized
portions allows the qualitative determination of the presence or absence of
PRE DNA.
Similarly, a Northern transfer can be used for the detection of RNA
containing PRE sequences. For example, RNA is isolated from a given cell
sample using an acid guanidinium-phenol-chloroform extraction method. The
RNA is then electrophoresed to separate different species and transferred
from the gel to a nitrocellulose membrane. As with the Southern transfers,
labeled probes or PCR can be used to identify the presence or absence of PRE
nucleic acid.
Sandwich assays are commercially useful hybridization assays for detecting
or isolating protein or nucleic acid. Such assays utilize a "capture"
nucleic acid or protein that is often covalently immobilized to a solid
support and a labeled "signal" nucleic acid, typically in solution. A
clinical or other sample provides the target nucleic acid or protein. The
"capture" nucleic acid or protein and "signal" nucleic acid or protein
hybridize with or bind to the target nucleic acid or protein to form a
"sandwich" hybridization complex. To be effective, the signal nucleic acid
or protein cannot hybridize or bind substantially with the capture nucleic
acid or protein.
Typically, oligonucleotide probes are labeled signal nucleic acids that are
used to detect hybridization. Complementary probe nucleic acids or signal
nucleic acids may be labeled by any one of several methods typically used to
detect the presence of hybridized polynucleotides. Methods of detection can
use labels for autoradiography or autofluorography, such as 3H,
125I, 35S, 14C, or 32P-labeled
probes or the like (see definition of label, above). Other labels include
ligands which bind to labeled antibodies, fluorophores, chemiluminescent
agents, enzymes, and antibodies which can serve as specific binding pair
members for a labeled ligand.
Detection of a hybridization complex may require the binding of a signal
generating complex to a duplex of target and probe polynucleotides or
nucleic acids. Typically, such binding occurs through ligand and anti-ligand
interactions as between a ligand-conjugated probe and an anti-ligand
conjugated with a signal, i.e., antibody-antigen or complementary nucleic
acid binding. The label may also allow indirect detection of the
hybridization complex. For example, where the label is a hapten or antigen,
the sample can be detected by using antibodies. In these systems, a signal
is generated by attaching fluorescent or enzymatic molecules to the
antibodies or, in some cases, by attachment of a radioactive label. The
sensitivity of the hybridization assays may be enhanced through use of a
target nucleic acid or signal amplification system which multiplies the
target nucleic acid or signal being detected. These systems can be used to
directly identify PRE variations, polymorphisms, or mutated sequences.
Alternatively, the specific sequences can be amplified using, e.g., generic
PCR primers, and the amplified target region later probed or sequenced to
identify a specific sequence indicative of the variant, polymorphism or
mutation.
Nucleic acid hybridization assays for the detection of isoforms, mutations
and for sequencing can also be performed in an array-based format. Arrays
are a multiplicity of different "probe" or "target" nucleic acids (or other
compounds) are hybridized against a target nucleic acid. In this manner a
large number of different hybridization reactions can be run essentially "in
parallel". This provides rapid, essentially simultaneous, evaluation of a
wide number of reactants. Methods of performing hybridization reactions for
detection and sequencing in array based formats are well known, e.g.,
Pastinen (1997) Genome Res. 7:606-614; Jackson (1996) Nature
Biotechnology 14:1685; Chee (1995) Science 274:610.
An alternative means for determining the level of expression of a gene is in
situ hybridization. In situ hybridization assays are well known (e.g.,
Angerer (1987) Methods Enzymol 152:649). In a typical in situ
hybridization assay, cells are fixed to a solid support, typically a glass
slide. If a nucleic acid is to be probed, the cells are typically denatured
with heat or alkali. The cells are then contacted with a hybridization
solution at a moderate temperature to permit annealing of labeled probes
specific to the nucleic acid sequence. The probes are typically labeled,
i.e., with radioisotopes or fluorescent reporters. Another well-known in
situ hybridization technique is the so-called fluorescence in situ
hybridization (FISH), see, e.g., Macechko (1997) J. Histochem. Cytochem.
45:359-363; Raap (1995) Hum. Mol. Genet. 4:529-534.
The sensitivity of the hybridization assays may be enhanced through use of a
nucleic acid amplification system which multiplies the target nucleic acid
being detected. Alternatively, the select sequences can be generally
amplified using nonspecific PCR primers and the amplified target region
later probed for a specific sequence indicative of a mutation.
Oligonucleotides for use as probes, e.g., in vitro amplification methods, as
gene probes in diagnostic methods, or as inhibitor components (see below)
are typically synthesized chemically; e.g., such as by the solid phase
phosphoramidite triester method described by Beaucage and Caruthers, supra,
or, using an automated synthesizer, as described in Needham-VanDevanter,
supra. Purification of oligonucleotides, where necessary, is typically
performed by native acrylamide gel electrophoresis or by anion-exchange HPLC
as described in Pearson and Regnier. The sequence of the synthetic
oligonucleotides can be verified using the chemical degradation method of
Maxam and Gilbert (Maxam (1980) supra).
It will be appreciated that nucleic acid hybridization assays can also be
performed in an array-based format. In this approach, arrays bearing a
multiplicity of different "probe" nucleic acids are hybridized against a
target nucleic acid. In this manner a large number of different
hybridization reactions can be run essentially "in parallel". This provides
rapid, essentially simultaneous, evaluation of a wide number of reactants.
Methods of performing hybridization reactions in array based formats are
well known to those of skill in the art (see. e.g., Jackson (1996) Nature
Biotechnol. 14:1685, and Chee (1995) Science 274:610).
6. Sequence Comparison Analysis
PRE-encoding nucleic acid sequences of the invention include both genes and
gene transcription products (mRNA) identified and characterized by analysis
of PRE sequences. Optimal alignment of sequences for comparison can be
conducted as described herein (see definitions). Sequence identity analysis
can also supplement functional analysis to determine whether a nucleic acid
is within scope of the invention. For example, in other embodiments, a PRE
sequence of the invention has at least 80% nucleic acid sequence identity to
SEQ ID NO:1, has at least 90% nucleic acid sequence identity to the sequence
as set forth in SEQ ID NO:1, or can comprise a sequence as set forth in SEQ
ID NO:1. Publicly available nucleic acid databanks can be searched for
sequence identity (homology) to the exemplary SEQ ID NO:1 PRE of the
invention to identify additional members of the PRE family of the invention.
Any of the programs described herein (see definitions) can be used to
identify PRE family members.
For example, the program PileUp was used to identify a PRE of the invention.
PILEUP creates a multiple sequence alignment from a group of related
sequences using progressive, pairwise alignments to show relationship and
percent sequence identity. It also plots a tree or dendogram showing the
clustering relationships used to create the alignment. PILEUP uses a
simplification of the progressive alignment method of Feng & Doolittle
(1987) supra; see also the method of Higgins & Sharp (1989) supra. The
program can align up to 300 sequences, each of a maximum length of 5,000
nucleotides or amino acids. The multiple alignment procedure begins with the
pairwise alignment of the two most similar sequences, producing a cluster of
two aligned sequences. This cluster is then aligned to the next most related
sequence or cluster of aligned sequences. Two clusters of sequences are
aligned by a simple extension of the pairwise alignment of two individual
sequences. The final alignment is achieved by a series of progressive,
pairwise alignments. The program is run by designating specific sequences
and their amino acid or nucleotide coordinates for regions of sequence
comparison and by designating the program parameters. For example, a
reference sequence can be compared to other test sequences to determine the
percent sequence identity relationship using the following parameters:
default gap weight (3.00), default gap length weight (0.10), and weighted
end gaps. See also Morrison (1997) supra, for the use of PILEUP.
PileUP was used with the parameters: symbol comparison table:
GenRunData:pileupdna.cmp, GapWeight:2, GapLength Weight:1 (see Example 1) to
identify a PRE of the invention. The following PRE sequence thus identified
is within the scope of the invention, having about 83% sequence identity to
SEQ ID NO:1: AGGAGTTGCA AGGCTAAGC X ACTGCACAGG AGAGG X TCTG CGG XX TATAA
CGACTTTCTC CTGGGAGATA AGTCATCTTG CATGAAGGTT CTATG X TCAT, where X is any
nucleotide (SEQ ID NO:6).
The BLAST program can also be used to identify a PRE family member by
sequence identity. For example, BLAST can uses as defaults a wordlength (W)
of 11, the BLOSUM62 scoring matrix (see Henikoff (1992) Proc. Natl. Acad.
Sci. USA 89:10915-10919) alignments (B) of 50, expectation (E) of 10,
M=5, N=-;4, and a comparison of both strands. The term BLAST refers to the
BLAST algorithm which performs a statistical analysis of the similarity
between two sequences; see, e.g., Karlin (1993) Proc. Natl. Acad. Sci.
USA 90:5873-5787. One measure of similarity provided by the BLAST
algorithm is the smallest sum probability (P(N)), which provides an
indication of the probability by which a match between two nucleotide or
amino acid sequences would occur by chance.
II. Functional Analyses: Measuring Levels of Viral Expression
Functional analysis can supplement sequence identity analysis to determine
whether a nucleic acid is within scope of the invention and to characterize
the level of attenuation effected by the PRE. For example, cell cultures or
activated PMBCs can be infected in vitro with PRE-containing recombinant
viruses of the invention. The level of attenuation can be determined by
measuring the amounts of total virion or virus product produced. Viral
products include polypeptides (e.g., p24gag) and transcription
products (mRNA message). Viral polypeptides can can be quantitated by e.g.,
antibody based assays, enzymatic assays, and the like. A variety of standard
protocols for detecting and measuring the expression of proteins using
either polyclonal or monoclonal antibodies specific for the protein are
known in the art. Examples include enzyme-linked immunosorbent assay
(ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).
These and other assays are described, e.g., in Hampton et al.,
Serological Methods a Laboratory Manual, APS Press, St Paul Minn.,
1990); Maddox (1983) J. Exp. Med. 158:121 1); Coligan, C
When the PRE-containing hybrid HIV-1 virus infects activated huPBMCs, the
level of expression of HIV-1 p24gag is between about 5 fold and
about 200 fold less than levels of p24gag expression when HIV-1
wild type virus infects activated huPBMCs. Levels of p24gag
expression can be measured by any means known in the art, such as, e.g.,
antibody based assays, as ELISA assays. For example, virus propagation can
be monitored over time using a p24gag antigen capture ELISA
assay. A commercial ELISA assay (Cellular Products, Buffalo, N.Y.) used
according to manufacturer's instructions or any p24gag antigen
capture assay using techniques well known in the art can be used (see, e.g.,
Zolotukhin (1994) supra; Van Doornum (1998) J. Med. Virol.
54:285-290; Hashida (1998) J. Clin. Lab. Anal. 12:115-120; Palenzuela
(1997) J. Immunol. Methods 208:43-48).
III. Mutagenesis of PRE Nucleic Acid
The invention also provides for PRE nucleic acid that have been modified in
a site-specific manner to modify, add to, or delete some or all functions.
For example, specific base pairs can be modified to alter, increase or
decrease the affinity of NCTE binding proteins, thus modifying the relative
level of attenuation. Alternatively, modifications can change the stability
of the secondary structure of the nucleic acid. Base pair changes can
augment expression of the nucleic acid in a cell, such as a bacteria.
Site-specific mutations can be introduced into PRE-encoding nucleic acid by
a variety of conventional techniques, well described in the scientific and
patent literature. Illustrative examples include: site-directed mutagenesis
by overlap extension polymerase chain reaction (OE-PCR), as in Urban (1997)
Nucleic Acids Res. 25:2227-2228; Ke (1997) Nucleic Acids Res
25:3371-3372; Chattopadhyay (1997) Biotechniques 22:1054-1056,
describing PCR-based site-directed mutagenesis "megaprimer" method; Bohnsack
(1997) Mol. Biotechnol. 7:181-188; Ailenberg (1997) Biotechniques
22:624-626, describing site-directed mutagenesis using a PCR-based
staggered re-annealing method without restriction enzymes; and Nicolas
(1997) Biotechniques 22:430-434, describing site-directed mutagenesis
using long primer-unique site elimination and exonuclease III.
Modified PRE of the invention can be further produced by chemical
modification methods, see, e.g., Belousov (1997) Nucleic Acids Res.
25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380;
Blommers (1994) Biochemistry 33:7886-7896.
IV. Expression of PRE Nucleic Acid
The invention provides for methods and reagents the expression of novel PRE
nucleic of the invention in any prokaryotic, eukaryotic, yeast, fungal,
plant, insect, human or animal cell. Antisense, in addition to sense,
sequences are provided. To create cell-based and in vitro assay systems to
screen for novel NCTEs using PRE of the invention, a variety of in vivo and
in vitro expression systems are provided.
A. Vectors and Transcriptional Control Elements
The invention provides for methods and reagents for expressing the novel PRE
of the invention as sense or antisense coding sequences, or in other
constructs, such as ribozymes. Other embodiments of the invention provide
methods and reagents for identifying, isolating and using PRE to identify
and isolate trans-acting NCTE binding proteins. After the coding region of a
PRE has been identified, it can be expressed by operably linking the coding
region to transcriptional regulatory elements, such as promoters and
enhancers. These sequences have characteristic subsequences, for instance,
promoter sequence elements typically include the TATA box consensus sequence
(TATAAT), which is usually 20 to 30 base pairs upstream of the transcription
start site. Promoters can be tissue-specific or not, constitutive or
inducible. Promoters that drive expression continuously under physiological
conditions are referred to herein as "constitutive" promoters and are active
under most environmental conditions and states of development or cell
differentiation. Typical expression systems, such as expression cassettes
and vectors, also contain transcription and translation terminators,
transcription and translation initiation sequences. Generic expression
cassettes typically contain at least one independent terminator sequence,
sequences permitting replication of the cassette in eukaryotes, or
prokaryotes, or both, (e.g., shuttle vectors) and selection markers for both
prokaryotic and eukaryotic systems. See, e.g., Roberts (1987) Nature
328:731; Berger (1987) supra; Schneider (1995) Protein Expr. Purif.
6435:10; Sambrook and Ausubel. Product information from manufacturers of
biological reagents and experimental equipment also provide biological
methodologies, such as, e.g., the SIGMA Chemical Company (Saint Louis, Mo.),
Pharmacia Biotech (Piscataway, N.J.), Clontech Laboratories, Inc. (Palo
Alto, Calif.), Aldrich Chemical Company (Milwaukee, Wis.), GIBCO BRL Life
Technologies, Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (Fluka
Chemie A G, Buchs, Switzerland). The promoters and vectors used in this
invention can be isolated from natural sources, obtained from such sources
as ATCC or GenBank libraries, or prepared by synthetic methods, as described
herein.
The PRE sequences of the invention can be expressed in cassettes or vectors
which are transiently expressed in cells using, e.g., episomal vectors such
as vaccinia virus, see Cooper (1997) Proc Natl Acad Sci USA
94:6450-6455. They can include sequences coding for episomal maintenance and
replication such that integration into the host genome is not required.
Alternatively, PRE coding sequences can be inserted into the host cell
genome becoming an integral part of the host chromosomal DNA, using, e.g.,
retroviral vectors such as SIV or HIV, see e.g., Naldini (1996) Science
272:263-267. Expression vectors can contain selection markers that
confer a selectable phenotype on transformed cells. For example, a marker
may encode antibiotic resistance, as to chloramphenicol, kanamycin, G418,
bleomycin or hygromycin, to permit selection of those cells transformed with
the desired DNA sequences, see, e.g., Blondelet-Rouault (1997) Gene
190:315-317. Because selectable marker genes conferring resistance to
substrates like neomycin or hygromycin can only be utilized in tissue
culture, chemoresistance genes are also used as selectable markers in vitro
and in vivo. Various target cells are rendered resistant to anticancer drugs
by transfer of chemoresistance genes encoding, e.g., P-glycoprotein,
multidrug resistance-associated protein-transporter, dihydrofolate reductase,
glutathione -S-transferase, O 6-alkylguanine DNA alkyltransferase, or
aldehyde reductase (Licht (1997) Stem Cells 15:104-111). Illustrative
vectors incorporating PRE of the invention include, e.g., adenovirus-based
vectors (Cantwell (1996) Blood 88:4676-4683; Ohashi (1997) Proc
Natl Acad Sci USA 94:1287-1292), Epstein-Barr virus-based vectors (Mazda
(1997) J Immunol Methods 204:143-151), adenovirus-associated virus
vectors, Sindbis virus vectors (Strong (1997) Gene Ther. 4: 624-627),
Herpes simplex virus vectors (Kennedy (1997) Brain 120: 1245-1259)
and retroviral vectors (Schubert (1997) Curr Eye Res 16:656-662).
Epstein-Barr virus episomal vectors (Horlick (1997) Protein Expr. Purif.
9:301-308, and plasmid DNA (Lowrie (1997) Vaccine 15: 834-838); all
of which can be used to express the nucleic acids of the invention in vivo
or ex vivo
V. Inhibiting Expression of PRE Nucleic Acid
The invention further provides for nucleic acids which can inhibit the
expression or function of PRE nucleic acids. These inhibitory nucleic acids
are typically complementary to, i.e., are antisense sequences to, the PRE of
the invention. Expression of inhibitory nucleic acid sequences can be used
to completely inhibit or further depress the replicative potential of an
attenuated virus. For example, a hybrid HIV-1 virus can be designed to
express inhibitory nucleic acid sequence under the control of an inducible
promoter. Thus, if desired, after administration of a PRE-attenuated viral
vaccine, the expression and function of the sense PRE, and thus the
replicative potential of the virus, can be down-regulated or turned off.
The inhibition can be effected through the targeting of genomic DNA or
messenger RNA. The transcription or function of targeted nucleic acid can be
inhibited, for example, by hybridization and/or cleavage. One particularly
useful set of inhibitors provided by the present invention includes
oligonucleotides which are able to either bind PRE gene or message, in
either case preventing or inhibiting the production, splicing, transport, or
function of viral message. The association can be though sequence specific
hybridization. Another useful class of inhibitors includes oligonucleotides
which cause inactivation or cleavage of PRE-containing message. The
oligonucleotide can have enzyme activity which causes such cleavage, such as
ribozymes. The oligonucleotide can be chemically modified or conjugated to
an enzyme or composition capable of cleaving the complementary nucleic acid.
One may screen a pool of many different such oligonucleotides for those with
the desired activity.
1. Antisense Oligonucleotides
The invention provides for with antisense oligonucleotides capable of
binding PREcontaining messagc to inhibit or further depress the replicative
potential of a PRE-containing attenuated virus. Strategies for designing
antisense oligonucleotides are well described in the scientific and patent
literature, and the skilled artisan can design such PRE complementary
oligonucleotides using the novel reagents of the invention. In some
situations, naturally occurring nucleic acids used as antisensc
oligonucleotides may need to be relatively long (18 to 40 nucleotides) and
present at high concentrations. A wide variety of synthetic, non-naturally
occurring nucleotide and nucleic acid analogues are known which can address
this potential problem. For example, peptide nucleic acids (PNAs) containing
non-ionic backbones, such as N-(2-aminoethyl) glycine units can be used.
Antisense oligonucleotides having phosphorothioate linkages can also be
used, as described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol Appl
Pharmacol 144:189-197; Antisense Therapeutics, ed. Agrawal (Humana
Press, Totowa, N.J., 1996). Antisense oligonucleotides having synthetic DNA
backbone analogues provided by the invention can also include
phosphoro-dithioate, methylphosphonate, phosphoramidate, alkyl
phosphotriester, sulfamate, 3′-thioacetal, methylene(methylimino), 3′-N-carbamate,
and morpholino carbamate nucleic acids, as described above.
Combinatorial chemistry methodology can be used to create vast numbers of
oligonucleotides that can be rapidly screened for specific oligonucleotides
that have appropriate binding affinities and specificities toward any
target, such as the sense and antisense PRE sequences of the invention (for
general background information, see, e.g., Gold (1995) J. of Biol. Chem.
270:13581-13584).
2. Inhibitory Ribozymes
The invention provides for with ribozymes capable of targeting
PRE-containing message to inhibit, e.g., the splicing, transport,
protein-binding capacity, or translation of viral mRNA, for further
attenuating a PRE-containing hybrid virus. Strategies for designing
ribozynies and selecting PRE antisense sequence for targeting are well
described in the scientific and patent literature, and the skilled artisan
can design such ribozynies using the novel reagents of the invention.
Ribozymes act by binding to a target RNA through the target RNA binding
portion of a ribozyme which is held in close proximity to an enzymatic
portion of the RNA that cleaves the target RNA. Thus, the ribozyme
recognizes and binds a target RNA through complementary base-pairing, and
once bound to the correct site, acts enzymatically to cleave and inactivate
the target RNA. For example, ribozyme cleavage of PRE-containing message
would prevent binding to NCTE binding protein, thus preventing subsequent
transport of the message to the cytoplasm. After a ribozyme has bound and
cleaved its RNA target, it is typically released from that RNA and so can
bind and cleave new targets repeatedly.
The effective concentration of ribozyme necessary to effect a therapeutic
treatment can be lower than that of an antisense oligonucleotide. This
potential advantage reflects the ability of the ribozyme to act
enzymatically. Thus, a single ribozyme molecule is able to cleave many
molecules of target RNA. In addition, a ribozyme is typically a highly
specific inhibitor, with the specificity of inhibition depending not only on
the base pairing mechanism of binding, but also on the mechanism by which
the molecule inhibits the expression of the RNA to which it binds. That is,
the inhibition is caused by cleavage of the RNA target and so specificity is
defined as the ratio of the rate of cleavage of the targeted RNA over the
rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent
upon factors additional to those involved in base pairing. Thus, the
specificity of action of a ribozyine can be greater than that of antisense
oligonucleotide binding the same RNA site.
The enzymatic ribozyme RNA molecule can be formed in a hammerhead motif, but
may also be formed in the motif of a hairpin, hepatitis delta virus, group I
intron or RNaseP-like RNA (in association with an RNA guide sequence).
Examples of such hammerhead motifs are described by Rossi (1992) Aids
Research and Human Retroviruses 8:183; hairpin motifs by Hampel (1989)
Biochemistry 28:4929, and Hampel (1990) Nuc. Acids Res.
18:299; the hepatitis delta virus motif by Perrotta (1992) Biochemistry
31:16; the RNaseP motif by Guerrier-Takada (1983) Cell 35:849;
and the group I intron by Cech U.S. Pat. No.4,987,071. The recitation of
these specific motifs is not intended to be limiting; those skilled in the
art will recognize that an enzymatic RNA molecule of this invention has a
specific substrate binding site complementary to one or more of the target
gene RNA regions, and has nucleotide sequence within or surrounding that
substrate binding site which imparts an RNA cleaving activity to the
molecule.
VI. Construction of Attenuated Virus and Viral Vaccine
The invention provides for an attenuated retrovirus and vaccine comprising
the PRE of the invention. One means to genetically engineer a wild-type,
virulent virus to a hybrid, attenuated virus involves constructing a virus
which either lacks or has a non-functional endogenous post-transcriptional
RNA nucleo-cytoplasmic transport elements (NCTEs). The endogenous NCTE is
subsequently replaced by the exogenous NCTE of the invention which functions
less efficiently in vivo than its wild-type counterpart, thus effecting the
attenuation. For example, insertion of the PRE of the invention in a RRE(-;)
and/or Rev(-;) HIV-1 creates a slower growing, "attenuated" hybrid virus.
This level of attenuation can be measured. When the PRE-containing hybrid
HIV-1 virus infects activated huPBMCs, the level of expression of HIV-1 p24gag
is between about 5 fold and about 200 fold less than levels of p24gag
expression when HIV-1 wild type virus, utilizing wild-type NCTE,
infects activated huPBMCs. Furthermore, in constructing the attenuated
retrovirus of the invention, additional elements of the retrovirus which are
essential for its replication and/or pathogenicity can also be disabled or
eliminated, such as Nef, as explained below.
In normal mammalian cells, message RNA, present in the cell as
ribonucleoprotein (RNP) complexes, is only exported from the nucleus to the
cytoplasm after splicing is completed. To circumvent the requirement of
splicing prior to export from the nucleus, all retroviruses have evolved a
mechanism that allows the nuclear export of unspliced form of viral RNAs
which are necessary for the production of structural proteins and essential
for viral replication. This mechanism involves the highly structured NCTE
cis-acting RNA element and its corresponding trans-acting RNA binding
proteins, as discussed above. In simian type retroviruses, the NCTE is
termed "CTE" (see Bray (1994) supra; Zolotukhin (1994) supra), and binds to
endogenous cellular RNA binding proteins. In contrast, HIV-1's NCTE does not
bind cellular NCTE-binding proteins. It encodes its own NCTE binding
protein, called "Rev." Rev interacts with a specific HIV-1 NCTE sequence,
designated the "Rev-responsive element," or "RRE," contained in its gag/pol
and env encoding transcript. Rev interacts directly with RRE as part of the
RNA export machinery which transports RRE-containing transcripts to the
cytoplasm from the nucleus. As a result, HIV-1 needs both RRE and Rev to
produce infectious virus. Disabling either produces a non-replicative,
non-virulent virus. Replacing (i.e., reconstituting) HIV-1's RRE/Rev RNA
transport mechanism with a less efficient NCTE, such as the PRE of the
invention, produces an attenuated and avirulent hybrid virus.
To engineer a non-functional RRE and/or Rev, the skilled artisan can delete
and or mutate any portion of the RRE or Rev coding sequence. Means to delete
or mutate nucleic acid sequence are described herein, and are well known in
the art. Construction of exemplary, attenuated retroviruses are also
discussed in the Examples, below. RRE and Rev sequences are well known in
the art, e.g., see databases, such as the NCBI database at http:/www.ncbi.nlm.nih.gov/Entrez/nucleotide.html
or http://www.ncbi.nlm.nih.gov/Entrez/protein.html. Further description and
sequence of HIV-1 Rev can be found in, e.g., Salminen (1997) J. Virol.
71:2647-2655, Accession U86770; Theodore (1996) AIDS Res. Hunt.
Retroviruses 12:191-194, Accession AF004394; Fang, et al., Accession
AF003887; Howard (1996) AIDS Res. Hum. Retroviruses 12:1413-1425,
Accession L39106; to name only a few. Further description and sequence of
HIV-1 RRE can be found in, e.g., Salminen (1996) JOURNAL AIDS Res. Hum.
Retroviruses 12:1329-1339, Accession U46016, WO 9202228-A5 20-FEB-1992,
Accession A20711; Battiste (1994) Biochemistry 33:2741-2747; Battiste
(1995) J. Biomol. NMR 6:375-389; Battiste (1996) Science
273:1547-1551; to name just a few.
The PRE of the invention can be inserted at any position in the disabled
(lacking an endogenous NCTE) retroviral genome as long as the insertion does
not inactivate the virus. The point of insertion can be designed or altered
to modify the level of attenuation for a given PRE. In one embodiment of the
invention, the PRE is inserted in the 3′ untranslated region of a disabled
retrovirus. In alternative embodiments, the PRE is inserted in the region of
the disabled NCTE sequence (e.g., RRE in HIV-1) or the Nef region. Each
potential point of insertion must be investigated individually for efficacy
and level of attenuation. For example, as described in Example 1, the PRE of
SEQ ID NO:1 has been inserted in the Nef region of a disabled HIV-1
construct, at nucleotide (nt) 8887 of pNL4-3 (as described in Example 1) to
successfully generate a PRE-attenuated virus. However, when the site of
insertion was at nt 8786, located between the env and nefgenes, the hybrid
failed to generate infectious virus. Thus, each insertion site must be
individually tested for its ability to accept a PRE sequence to generate an
infectious and non-pathogenic hybrid.
To further engineer and modify a desired levels of nucleo-cytoplasmic
transport, message stability, rate of virion growth, levels of attenuation,
and the like, more that one PRE, or different PREs, or both, can be inserted
into a given retroviral construct.
In constructing the attenuated retroviruses and vaccines of the invention,
in addition to endogenous NCTE, other elements essential for the virus'
replication and/or pathogenicity can also be disabled or eliminated. For
example, genetic engineering of a Nef-negative retrovirus may produce a
recombinant hybrid with a greater degree of attenuation. In the case of
HIV-1, a functional Nef gene is important for development of high viremia
and AIDS. Animals infected with Nef-deleted attenuated viruses are resistant
to subsequent challenge with pathogenic wild-type viruses. Some individuals
with long-term nonprogressive HIV-1 infection (no clinical or immunological
signs of immuno-deficiency despite being HIV seropositive for over a decade)
are infected with viruses having naturally occurring Nef deletions. To
engineer a non-functional Nef, the skilled artisan can delete and or mutate
any portion of the Nef coding sequence. Nef sequences are well known in the
art, e.g., see databases, such as the NCBI databases described above. For
examples of HIV-1 Nef nucleic acid and polypeptide sequences, see, e.g.,
Accession Nos. Y15123, U88826, Y15121, Y15120, Y15116, to name only a few.
For a further description of Nef, see, e.g., Saksela (1997) supra; Greenberg
(1997) supra; Luo (1997) J. Virol. 71:9531-9537; Luo (1997) J.
Virol. 71:9524-9530; Okada (1997) supra.
VII. Delivery of Nucleotides into Cells
The nucleic acids and oligonucleotides of the invention, including
expression cassettes and vectors expressing PRE, can be delivered into cells
in culture, tissues and organisms for synthesis, mutation, screening and the
like. For example, the invention provides for a method for screening for a
post-transcriptional RNA nucleo-cytoplasmic transport element (NCTE) binding
protein. The method involves contacting a PRE of the invention with a test
compound and measuring the ability of the test compound to bind the NCTE.
This screening technique can be used in intact cells. Inhibitory
oligonucleotides of the invention, and vectors capable of expressing these
sequences, are also transferred into intact cells in cell culture, tissues
or intact organisms.
The nucleic acids and oligonucleotides of the invention can be transferred
into a cell using a variety of techniques well known in the art. For
example, oligonucleotides can be delivered into the cytoplasm spontaneously,
without specific modification. Altematively, they can be delivered by the
use of liposomes which fuse with the cellular membrane or are endocytosed,
i.e., by employing ligands attached to the liposome, or attached directly to
the oligonucleotide, that bind to surface membrane protein receptors of the
cell resulting in endocytosis. For example, a DNA binding protein, e.g.,
HBGF-1, is known to transport oligonucleotides into a cell. See, e.g., Tseng
(1997) J. Biol. Chem. 272:25641-25647; Satoh (1997) Biochem.
Biophys. Res. Commun. 238:795-799, describing efficient gene
transduction by Epstein-Barr-virus-based vectors coupled with cationic
liposome and HVJ-liposome. Displaying ligands specific for target cells on
the surface of a liposome targets the construct to a specific cell or organ
in vivo. See, e.g., Huwyler (1997) J. Pharmacol. Exp. Ther.
282:1541-1546, describing receptor mediated delivery using immunoliposomes.
Cells can also be permeabilized to enhance transport of oligonucleotides
into the cell, without injuring the host cells. See, e.g., Verspohl (1997)
Cell. Biochem. Funct. 15:127-134; Kang (1997) Pharm. Res.
14:706-712; Bashford (1994) Methods Mol. Biol. 27, 295-305,
describing use of bacterial toxins for membrane permeabilization; and for
general principles of membrane permeabilization, see, e.g., Hapala (1997)
Crit. Rev. Biotechnol. 17:105-122.
VIII. Preparation, Formulation and Administration of Attenuated Viral
Vaccines
Live PRE-attenuated retrovirus, such as HIV-1, can be grown and harvested
from activated human peripheral mononuclear cells or from a variety of
tissue culture cells, such as human 293 cell line, as described herein; see
also, e.g., Eberlein (1991) Virus Res. 19:153-161; Parente (1996)
Gene Ther. 3:756-760; Margolis (1997) AIDS Res. Hum. Retroviruses
13:1411-1420. Virion-containing supernatants are collected, and, typically,
filtered. PRE sequences in the harvested, attenuated virus for use in
vaccine formulations can be confirmed by conventional sequencing. The
attenuated virus can be further purified, e.g., by ultrafiltration or
ultra-centrifugation. The live, attenuated virus can be stored, e.g., by
refrigeration, or on a long-term basis, by freezing in liquid nitrogen.
A formulation for administering the virus as a vaccine is prepared using,
e.g., any physiologically acceptable buffer, such as saline or phosphate
buffered saline (PBS). This can be stored in a frozen state. The formulation
can also be freeze-dried, stored at room temperature, and reconstituted by
adding appropriate volume of buffer. The vaccine pharmaceutical formulation
can be in the form of a sterile injectable preparation, such as a sterile
injectable aqueous or oleaginous suspension. This suspension can be
formulated according to the known art using those suitable dispersing or
wetting agents and suspending agents which have been mentioned above. The
sterile injectable preparation can also be a solution or suspension in a
nontoxic parenterally-acceptable diluent or solvent, such as a solution of
1,3-butanediol. Among the acceptable vehicles and solvents that can be
employed are water and Ringer's solution, an isotonic sodium chloride. In
addition, sterile fixed oils can conventionally be 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 can likewise be used in the preparation of injectables.
The PRE-containing attenuated virus and vaccine formulations can also be
co-administered with other reagents to boost or otherwise augment the
anti-viral immune response. For example, they can be administered with an
adjuvant, e.g., oil-in-water microemulsions or polymeric microparticles.
Oil-in-water microemulsions are potent and safe adjuvants in humans and have
been used with HSV-2, HIV-1 and influenza virus vaccines. Microparticles can
be prepared from the biodegradable polymers, e.g.,
poly(lactide-co-glycolides). See, e.g., O'Hagan (1998) J. Pharm Pharmacol.
50:1-10.
The live attenuated viral vaccine of the invention can be administered using
any acceptable route, as, e.g., by application to a mucosal surface, by
injection, by inhalation (such as by aerosol) or other intranasal route, or
by ingestion. For examples of inhalants, see Rohatagi (1995) J. Clin.
Pharmacol. 35:1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol.
75:107-111; Fernandez-de Castro (1997) Salud Publica Mex 39:53-60.
Injection of vaccine can be intravenous or intramuscular; see, e.g.,
Groswasser (1997) Pediatrics 100:400-403, as example of injection
techniques for efficient intramuscular vaccine delivery. Administration by
application to any mucosal surface, including, e.g., intraoral (sublingual,
buccal, and the like), intranasal, intrarectal, intravaginal, or ocular. For
examples of mucosal administration methods, see, e.g., Staats (1997) AIDS
Res Hum Retroviruses 13:945-952; Okada (1997) J. Immunol.
159:3638-3647; Wu (1997) AIDS Res Hum Retroviruses 13:1187-1194.
The amount of virus (number of virions) per dose will vary depending on
results of different titrations used in clinical trials. The range can
range, e.g., from only a few infectious units, to about 104 to 1010
infectious units (i.e., virions) per dose. Protocols and means to
determine safety and efficacy used for other attenuated vaccines can be
adapted and used with the novel reagents provided by the invention; see,
e.g., Beishe (1998) N. Engl. J. Med. 338:1405-1412; Gruber (1997)
Vaccine 15:1379-1384; Tingle (1997) Lancet 349:1277-1281; Varis
(1996) J. Infect. Dis. 174:S330-S334; Gruber (1996) J. Infect. Dis.
173:1313-1319.
After the vaccine has formulated in an acceptable carrier, it can be placed
in an appropriate container and labeled. For administration of the vaccine,
such labeling would include, e.g., instructions concerning the amount
frequency and method of administration. In one embodiment, the invention
provides for a kit and instructional material teaching the indications,
dosage and schedule of administration of the vaccine.
Selection of individuals who would benefit from receiving the live,
attenuated vaccine of the invention include, but are not limited to,
individuals who have a high risk of being exposed to HIV, such as
intravenous drug users, individuals who may been exposed, as through a
needle stick or transfusion, and individuals whose exposure to the virus has
been confirmed, e.g., by a positive blood test.
The vaccine can be administered in conjunction with other treatment
regimens, e.g., it can be coadministered or administered before or after any
anti-viral pharmaceutical (see, e.g., Moyle (1998) Drugs 55:383-404)
or a killed (completely inactivated) anti-HIV vaccine. The vaccine can be
administered in any form of schedule regimen, e.g., in a single dose, or,
using several doses (e.g., boosters) at dosages and time intervals to be
determined by clinical trials.
The attenuated vaccine of the invention is considered efficacious, i.e.,
immunoprotective, if it elicits any protective or ameliorative humoral or
cell-mediated anti-HIV response. Preferably, the vaccine of the invention
will cause no side effects, clinically significant pathology, acceleration
of onset of symptoms, further dissemination of virus in the body, and the
like. The anti-HIV response can be assessed by any parameter, e.g., by
measuring the levels of anti-viral antibodies or HIV-specific T cells, the
amount of HIV virion or nucleic acid in the blood or lymph nodes (see, e.g.,
Brown (1997) Transfusion 37:926-929), the levels of circulating
helper (CD4+) T cells, and the like. See also, O'Brien (1997)
"Changes in plasma HIV RNA levels and CD4+ lymphocyte counts
predict both response to antiretroviral therapy and therapeutic failure,"
Ann. Intern. Med. 126:939-945; Hughes (1997) "Monitoring plasma HIV-1
RNA levels in addition to CD4+ lymphocyte count improves
assessment of antiretroviral therapeutic response," Ann. Intern. Med.
126:929-938; Burgisser (1997) "Monitoring responses to antiretroviral
treatment in human immunodeficiency virus type 1 (HIV-1)-infected patients
by serial lymph node aspiration," J. Infect. Dis. 175:1202-1205.
IX. Screening for NCTE Binding Proteins Using PRE
The invention provides for cell-based and in vitro assay systems to screen
for novel NCTE-binding proteins using the PRE of the invention. The
full-length PRE can be utilized, or, alternatively, a portion of a PRE can
be used to assay for NCTE binding proteins. One embodiment of the invention
provides for a method of screening for an NCTE binding protein by contacting
a PRE of the invention with a test compound and measuring the ability of the
test compound to bind the NCTE. Many assays are available that screen for
nucleic acid binding proteins and all can be adapted and used with the novel
reagents provided for by the invention. A few illustrative example are set
forth below.
A variety of well-known techniques can be used to identify polypeptides
which specifically bind to nucleic acids, such as PRE. For example, mobility
shift DNA-binding assays, methylation and uracil interference assays, DNase
and hydroxy radical footprinting analysis, fluorescence polarization, and UV
crosslinking or chemical cross-linkers, can be used. For a general overview
of protein-nucleic acid binding assays, see, e.g., Ausubel (chapter 12,
DNA-Protein Interactions).
One technique for isolating co-associating proteins, including nucleic acid
and DNA/RNA binding proteins, includes use of UV crosslinking or chemical
cross-linkers, including, e.g., cleavable cross-linkers
dithiobis(succinimidylpropionate) and 3,3′-dithiobis (sulfosuccinimidyl-propionate);
see, e.g., McLaughlin (1996) Ann. J. Hum. Genet. 59:561-569, Tang
(1996) Biochemistry 35:8216-8225; Lingner (1996) Proc. Natl. Aca.
Sci. U.S.A. 93:10712; Chodosh (1986) Mol. Cell. Biol 6:4723-4733.
If a specific protein is believed to bind to PRE, and an antibody is
available or can be generated for that protein, co-immunoprecipitation
analysis can be used. Alternatively, PRE-affinity columns can be generated
to screen for potential PRE-binding proteins. In a variation of this assay,
PRE-containing nucleic acid is biotinylated, reacted with a solution
suspected of containing a PRE-binding protein, and then reacted with a
strepavidin affinity column to isolate the PRE-containing nucleic
acid/binding protein complex (see, e.g., Grabowski (1986) Science
233:1294-1299; Chodosh (1986) supra). The protein can then be conventionally
eluted and isolated.
Mobility shift DNA-protein binding assay using nondenaturing polyacrylarnide
gel electrophoresis is an extremely rapid and sensitive method for detecting
polypeptides binding to DNA (see, e.g., Chodosh (1986) supra, Carthew (1985)
Cell 43:439-448; Trejo (1997) J. Biol. Chem. 272:27411-27421;
Bayliss (1997) Nucleic Acids Res. 25:3984-3990). Interference assays
and DNase and hydroxy radical footprinting can be used to identify specific
residues in the nucleic acid protein-binding site, see, e.g., Bi (1997)
J. Biol. Chem. 272:26562-26572; Karaoglu (1991) Nucleic Acids Res.
19:5293-5300. Fluorescence polarization is a powerful technique for
characterizing macromolecular associations and can provide equilibrium
determinations of protein-DNA and protein-protein interactions. This
technique is particularly useful to study low affinity protein-protein
interactions, see, e.g., Lundblad (1996) Mol. Endocrinol. 10:607-612.
Proteins identified in by these techniques can be further separated on the
basis of their size, net surface charge, hydrophobicity and affinity for
ligands. In addition, antibodies raised against such proteins can be
conjugated to column matrices and the proteins immunopurified. All of these
general methods are well known in the art. See, e.g., Scopes, R. K., Protein
Purification: Principles and Practice, 2nd ed., Springer Verlag, (1987).
Chromatographic techniques can be performed at any scale and using equipment
from many different manufacturers (e.g., Pharmacia Biotech).
Claim 1 of 30 Claims
1. An isolated post-transcriptional regulatory element (PRE) nucleic acid
comprising SEQ ID NO:1, the PRE nucleic acid defined as having the
following property:
the PRE nucleic acid, when inserted in a recombinant, hybrid human
immunodeficiency virus (HIV)-1 lacking or having a non-functional
wild-type post-transcriptional RNA nucleo-cytoplasrnic transport element (NCTE),
functions as a NCTE in the hybrid HIV-1, and when the PRE-containing
hybrid HIV-1 virus infects activated human peripheral blood mononuclear
cells (huPBMCs), the level of expression of HIV-1 p24gag is
between about 5 fold and about 200 fold less than levels of p24gag
expression when HIV-1 wild type virus, utilizing wild-type NCTE,
infects activated huPBMCs.
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