U.S. patent application number 11/367657 was filed with the patent office on 2006-08-10 for lentiviral ltr-deleted vector.
This patent application is currently assigned to Oxford Biomedica (UK) Limited. Invention is credited to Alan John Kingsman, Susan Mary Kingsman.
Application Number | 20060177934 11/367657 |
Document ID | / |
Family ID | 26310305 |
Filed Date | 2006-08-10 |
United States Patent
Application |
20060177934 |
Kind Code |
A1 |
Kingsman; Alan John ; et
al. |
August 10, 2006 |
Lentiviral LTR-deleted vector
Abstract
A vector capable of transducing non-dividing and/or slowly
dividing cells is provided, wherein the vector is a lentiviral
LTR-deleted vector. Also provided is a method for producing a
protein of interest in a non-dividing or slowly dividing cell by
transducing the cell with a lentiviral LTR-deleted vector and
expressing the protein of interest in the cell. In addition, target
cells containing the lentiviral LIR-deleted vector are
provided.
Inventors: |
Kingsman; Alan John;
(Appleton, GB) ; Kingsman; Susan Mary; (Appleton,
GB) |
Correspondence
Address: |
FROMMER LAWRENCE & HAUG
745 FIFTH AVENUE- 10TH FL.
NEW YORK
NY
10151
US
|
Assignee: |
Oxford Biomedica (UK)
Limited
|
Family ID: |
26310305 |
Appl. No.: |
11/367657 |
Filed: |
March 3, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11155043 |
Jun 17, 2005 |
7056699 |
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11367657 |
Mar 3, 2006 |
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10324616 |
Dec 20, 2002 |
6924123 |
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11155043 |
Jun 17, 2005 |
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09254832 |
Jun 21, 1999 |
6541219 |
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PCT/GB97/02969 |
Oct 28, 1997 |
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10324616 |
Dec 20, 2002 |
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Current U.S.
Class: |
435/456 ;
435/368 |
Current CPC
Class: |
C12N 2799/027 20130101;
C12N 15/62 20130101; A61K 48/00 20130101; C12N 9/0071 20130101;
C12N 2830/48 20130101; C07K 2319/00 20130101; C12N 9/88 20130101;
C12N 2830/00 20130101; C12N 2830/15 20130101; C12N 2830/60
20130101; C12N 2740/16043 20130101; C12N 2830/50 20130101; C12N
2740/15043 20130101; C12N 15/86 20130101 |
Class at
Publication: |
435/456 ;
435/368 |
International
Class: |
C12N 15/867 20060101
C12N015/867; C12N 5/08 20060101 C12N005/08 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 29, 1996 |
GB |
9622500.8 |
Claims
1. A lentiviral vector capable of transducing a non-dividing or
slowly-dividing cell, said vector comprising a lentiviral
LTR-deleted vector.
2. The vector according to claim 1, further comprising a nucleotide
sequence encoding a protein of interest.
3. A method for producing a protein of interest in a non-dividing
or slowly-dividing cell, comprising the steps of: a) transducing
the cell with the vector according to claim 2; and b) expressing
the protein of interest in the cell.
4. The method according to claim 3, wherein the non-dividing cell
is a neuron.
5. A target cell in vitro comprising the vector of claim 1.
6. A target cell in vitro comprising the vector of claim 2.
7. A method of performing gene delivery on a target cell comprising
the steps of: a) transducing the target cell with the vector
according to claim 2; and b) delivering the nucleotide sequence to
the target cell.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 09/254,832, filed on Jun. 21, 1999, as the
national phase application of International application Serial No.
PCT/GB97/02969, filed on Oct. 28, 1997 and claiming priority to UK
application Serial No. GB 9622500.8, filed on Oct. 29, 1996. This
application makes reference to U.S. Pat. No. 6,235,522, filed on
Apr. 5, 1999 as the national phase application of International
application Serial No. PCT/GB97/02858, filed on Oct. 17, 1997 and
claiming priority to UK application Serial No. GB 9621680. This
application also makes reference to: U.S. Pat. No. 6,096,538, filed
on Nov. 19, 1997, U.S. Pat. No. 6,132,731, filed on Oct. 8, 1997,
U.S. Pat. No. 6,168,916, filed on Oct. 21, 1998, U.S. Pat. No.
6,312,682, filed on Dec. 28, 1998, U.S. Pat. No. 6,312,683, filed
on Jan. 27, 1999, U.S. application Ser. No. 09/533,276, filed on
Mar. 22, 2000, U.S. application Ser. No. 09/533,295, filed on Mar.
22, 2000, U.S. application Ser. No. 09/552,950, filed on Apr. 20,
2000, U.S. application Ser. No. 09/860,996, filed on May 18, 2001,
U.S. application Ser. No. 09/867,947, filed on May 29, 2001, U.S.
application Ser. No. 09/915,169, filed on Jul. 25, 2001, U.S.
application Ser. No. 10/001,220, filed on Nov. 15, 2001, U.S.
application Ser. No. 10/002,598, filed on Nov. 15, 2001, and U.S.
application Ser. No. 10/008,610, filed on Nov. 8, 2001.
[0002] Each document cited or referenced in each of the foregoing
applications, and any manufacturer's instructions or catalogues for
any products cited or mentioned in each of the foregoing
applications and in any of the cited documents, are hereby
incorporated herein by reference. Furthermore, all documents cited
in this text, all documents cited or referenced in documents cited
in this text, and any manufacturer's instructions or catalogues
fanny products cited or mentioned in this text or in any document
incorporated into this text, are incorporated herein by reference.
Documents incorporated by reference into this text or any teachings
therein can be used in the practice of this invention. Documents
incorporated by reference into this text are not admitted to be
prior art.
FIELD OF THE INVENTION
[0003] This invention relates to lentiviral long terminal repeat
(LTR)-deleted vectors. The invention also relates to lentiviral
LTR-deleted vectors carrying nucleotide sequences of interest, and
to their use in transferring genetic material to non-dividing or
slowly dividing
BACKGROUND OF THE INVENTION
[0004] Amongst nucleic acid transfer systems, retroviral vectors
hold substantial promise for gene therapy and other applications in
which transfer of genetic material is desirable. These systems can
transfer genes efficiently, and new vectors are emerging that are
particularly useful for gene delivery to brain cells (Naldini et
al., 1996 Science 272, 263).
[0005] There has been considerable interest in the development of
retroviral vector systems based on lentiviruses, a small subgroup
of the retroviruses. This interest arises firstly from the notion
of using HIV-based vectors to target anti-HIV therapeutic genes to
HIV susceptible cells and secondly from the prediction that,
because lentiviruses are able to infect non-dividing cells (Lewis
& Emerman 1993 J. Virol. 68, 510), vector systems based on
these viruses are able to transduce non-dividing cells (e.g. Vile
& Russel 1995 Brit. Med. Bull. 51, 12). Vector systems based on
HIV have been produced (Buchschacher & Panganiban 1992 J.
Virol. 66, 2731) and have been used to transduce CD4+ cells and
non-diving cells (Naldini et al., 1996 Science 272, 263). However,
in general, nucleic acid transfer efficiencies are not as high as
with comparable murine retrovirus vector systems.
[0006] The HIV-based vectors produced to date result in an
integrated provirus in the transduced cell that has HIV LTRs at its
ends. This limits the use of these vectors as the LTRs have to be
used as expression signals for any inserted gene unless an internal
promoter is used. The use of internal promoters has significant
disadvantages. For example, the presence of internal promoters can
affect the transduction titres obtainable from a packaging cell
line and the stability of the integrated vector.
[0007] Also, HIV and other lentiviral LTRs have virus-specific
requirements for nucleic acid expression. For example, the HIV LTR
is not active in the absence of the viral Tat protein (Cullen 1995
AIDS 9, S19). It is desirable, therefore, to modify or delete the
LTRs in such a way as to change the requirements for nucleic acid
expression. In particular, tissue specific gene expression signals
may be required for some gene therapy applications. In addition,
signals that respond to exogenous signals may be necessary. In
murine retroviruses this is often achieved simply by replacing the
enhancer-like elements in the U3 region of the murine lentiviral
(MLV) LTR by enhancers that respond to the desired signals. This
has not been feasible with viruses such as HIV because within the
U3 and R regions of their LTRs are sequences, known as IST and TAR,
which may inhibit gene expression and may or may not be responsive
to Tat protein when heterologous, perhaps tissue specific, control
sequences are inserted in the U3 region (Cullen 1995 AIDS 9, S19;
Alonso et al., 1994 J. Virol. 68, 6505; Ratnasabapathy et al., 1990
4, 2061; Sengupta et al., 1996 PNAS 87, 7492; Parkin et al., 1988
EMBO. J 7, 2831). Even if the signals are responsive, it is
undesirable to have to supply Tat as it further complicates the
system and Tat has some properties of oncoproteins (Vogel et al.,
1988 Nature 335, 606).
[0008] Parkinson's disease (PD) is a common neurodegenerative
disorder that afflicts the growing population of elderly people.
Patients display tremor, cogwheel rigidity and impairment of
movement. It is generally thought to be an acquired rather than
inherited disease in which environmental toxins, metabolic
disorders, infectious agents and normal aging have all been
implicated. PD is associated with the degeneration of nigrostriatal
neurons which have their soma located in the substantia nigra. They
send axonal projections to the basal ganglia and they use dopamine
as their neurotransmitter. Some features of the disease can be
controlled by the administration of L-DOPA, the metabolic precursor
to dopamine, which diffuses across the blood brain barrier more
effectively than dopamine itself. Unfortunately as the disease
progresses the side effects of this treatment become
unacceptable.
[0009] PD is an ideal candidate for gene therapy for several
reasons. The clinical efficacy of systemic administration of L-DOPA
suggests that restoration of neuronal circuitry is not essential
for disease management. Therefore genetic manipulation of brain
cells to provide local production of L-DOPA from tyrosine may be a
realistic strategy for treatment. The biosynthesis of L-DOPA from
tyrosine involves a single step suggesting that provision of
tyrosine hydroxylase (TH) by genetic means may be sufficient and
some success has been achieved using this strategy in small animals
and in cell culture (Kaplitt et al., 1994 Nature Genetics 8, 148;
During et al., 1994 Science 266, 1399; Horellou et al., 1994
Neuroreport 6, 49; Owens et al., 1991 J. Neurochem. 56, 1030).
However, if one is to use local endogenous brain cells as L-DOPA
factories for the treatment of PD in man it is likely that high
levels of L-DOPA will be required to effect a treatment. These high
levels must be efficiently converted to dopamine as the necessary
neurotransmitter and primary therapeutic agent. It is likely
therefore that it will be necessary not only to supply tyrosine
hydroxylase but also DOPA decarboxylase (DD), the enzyme that
converts L-DOPA to dopamine. This means that in a gene therapy
strategy the genes for both of these enzymes will be required.
However, it is clear from the literature that retroviral vectors
achieve the highest titres and most potent gene expression
properties if they are kept genetically simple (PCT/GB96/01230;
Bowtell et al., 1988 J. Virol. 62, 2464; Correll et al., 1994 Blood
84, 1812; Emerman and Temin 1984 Cell 39, 459; Ghattas et al., 1991
Mol. CeII. Biol. 11, 5848; Hantzopoulos et al., 1989 PNAS 86, 3519;
Hatzoglou et al., 1991 J. BioL Chem 266, 8416; Hatzoglou et al.,
1988 J. BioI. Chem 263, 17798; Li et al., 1992 Hum. Gen. Ther. 3,
381; McLachlin et al., 1993 Virol. 195, 1; Overell et al., 1988
MoI. Cell Biol. 8, 1803; Scharfman et al., 1991 PNAS 88, 4626; Vile
et al., 1994 Gene Ther 1, 307; Xu et al., 1989 Virol. 171, 331; Yee
et al., 1987 PNAS 84, 5197). This means only using a single
transcription unit within the vector genome and orchestrating
appropriate nucleic acid expression from sequences within the 5'
LTR. The need to express two enzymes from a single retroviral
vector would require the use of an internal ribosome entry site
(IRES) to initiate translation of the second coding sequence in a
poly-cistronic message (Adam et al. 1991 J. Virol. 65, 4985).
However, the efficiency of an IRES is often low and tissue
dependent making this strategy undesirable when one is seeking to
maximise the efficiency of metabolic conversion of tyrosine through
to dopamine. The present invention addresses these problems.
SUMMARY OF THE INVENTION
[0010] The present invention provides in one aspect a lentiviral
vector capable of transducing a non-dividing or slowly-dividing
cell, said vector comprising a lentiviral LTR-deleted vector. The
vector can further comprise a nucleotide sequence encoding a
polypeptide or protein of interest (POI), e.g., at least one
nucleotide sequence of interest (NOI) encoding at least one POI.
Advantageously, the NOI is operably linked to a promoter. If there
is more than one NOI, there can be one promoter for driving
expression, or a promoter for each NOI for driving expression.
Thus, one or more NOI can be operably linked to one or more NOI.
The vector can comprise a polynucleotide sequence, which encodes
two or more POI, e.g., therapeutic POI, operably linked to a
promoter, and the polynucleotide can encode a fusion POI. The
invention thus can provide a way of expressing two therapeutic NOI
from a single "chimeric" gene or polynucleotide. The vector may be
for example an expression vector such as a plasmid, or it may be a
retroviral vector particle comprising an RNA genome containing the
nucleotide sequences as described herein.
[0011] In another aspect, the invention provides a method for
producing a POI in a non-dividing or slowly-dividing cell,
comprising transducing the cell with a lentiviral LTR-deleted
vector and expressing the POI in the cell. In a preferred
embodiment, the non-dividing cell is a neuron.
[0012] There are many uses for in vitro expressed POI. For
instance, depending on the nature of the POI the in vitro expressed
POI can represent a protein that is purer than if the POI was
isolated from its native environment, as it would be free from
contaminants from that environment. Thus, such POI can be used in
assays, to generate antibodies, e.g. for use in assays, as antigens
or epitopes in immunological compositions, and as active agents in
therapeutic, pharmaceutical or veterinary compositions, inter
alia.
[0013] The invention further provides a target cell in vitro
comprising a lentiviral LTR-deleted vector. In yet further aspects,
the invention provides a DNA construct encoding the RNA genome for
the retroviral vector particle; and a retroviral vector production
system comprising a set of nucleic acid sequences encoding the
components of the retroviral vector particle.
[0014] The invention further provides the use of retroviral vectors
carrying the chimeric gene described herein, in gene therapy and in
the preparation of a medicament for gene therapy; and a method of
performing gene therapy on a target cell, which method comprises
transducing the target cell with a lentiviral LTR-deleted vector
comprising a nucleotide sequence encoding a POI, thus delivering
the nucleotide sequence to the target cell. The invention further
provides transduced target cells resulting from these methods and
uses. The invention thus provides a gene delivery system for use in
medicine.
[0015] The term "comprising" in this disclosure can mean
"including" or can have the meaning commonly given to the term
"comprising" in U.S. Patent Law.
[0016] Other aspects of the invention are described in or are
obvious from (and within the ambit of the invention) the following
disclosure.
BRIEF DESCRIPTION OF DRAWINGS
[0017] The following Detailed Description, given by way of example;
but not intended to limit the invention to specific embodiments
described, may be understood in conjunction with the accompanying
drawings, incorporated herein by reference, in which:
[0018] FIG. 1 shows a general scheme for Lentiviral LTR-deleted
(LLD) vectors which may be used with the present invention and
which are employed in the Examples.
[0019] FIG. 2 shows a generalised HIV-based LLD vector genome as
described in the Examples; Superscript H=HIV-derived sequence
(could be from any lentivirus); Superscript M=MLV-derived sequence;
1V=Packaging site (including gag region); PBS=Second strand priming
site; INTERNAL=Region containing genes, selectable markers, other
promoters or RNA handling systems such as HIV RRE and Rev coding
sequences.
[0020] FIG. 3 shows a specific HIV-based LLD vector genome as
described in the Examples. NIT vector genome (Inserts 3789
bp+backbone 2929 bp=6718 bp): HCMV promoter (-521 to -1) from
pRV109; HIV sequences (552 to 1144; 5861 to 6403; 7621 to 9085)
from HXB2; geonotype: gag-; pol-; env-; rev+; RRE; vif-; vpu-;
vpr-; tat-; nef-; mutations; three point mutations to remove ATG
(790, 834, 894) (@) a frameshift mutation by two base insertion
(831) (*); a deletion between NdeI (6403) and BgIII (7621)
(.DELTA.); polycloning site (X);
XhoI-SalI-ClaI-EcoRV-EcoRI-PstI-SmaI-SmaI-BamHI-SpeI (underlined
sites are unique); maximal insertion size into the polycloning
site: 5997 bp; backbone: pBluescriptKS+
[0021] FIG. 4 shows in detail the structure of the 3' LTR for the
vector in FIG. 3.
[0022] FIG. 5 shows a schematic diagram of packaging components
suitable for use with the vector genome shown in FIGS. 1 to 3.
pRV664 encodes HIV-1 HXB2 gagpol (637-5748) and contains RRE
(77208054) and its backbone is pCl-neo (PROMEGA). pRV438 possesses
both rev and env from HXB2 (5955-8902) in pSA91 which is a
mammalian expression plasmid with CMC promoter. pSyngp 160 nm (from
B. Seed) is an expression plasmid for HIV-1 MN envelope which was
modified to have the optimized codon usage in mammalian cells.
pRV67 is a VSV G expression plasmid in pSA91.
[0023] FIG. 6 further shows the principle of vectors according to
this invention.
[0024] FIG. 7 shows simplified directions for construction of
polynucleotide sequences according to the invention, encoding TH-DD
fusion proteins.
[0025] FIG. 8 shows simplified directions for construction of
polynucleotide sequences according to the invention, encoding DD-TH
fusion proteins.
[0026] FIG. 9 shows primers for use in the construction methods
illustrated in FIGS. 7 and 8 and described in detail in the
Examples. Lower case nucleotides denote tare codons in highly
expressed genes in mammalian cells (Haas et al., 1996 Cur Biol. 6,
315).
DETAILED DESCRIPTION OF THE INVENTION
[0027] The lentivirus of the invention provides the ability to
infect and transduce non-dividing and/or slowly-dividing cells.
During the infection process, lentiviruses form a pre-integration
complex in the target cell cytoplasm containing integrase, core
proteins and the proviral DNA. The complex is able to pass across
the nuclear membrane of the target cell, by means of signal
sequences in the proteins Other retroviruses either lack the
proteins, or have the proteins but without the appropriate signal
sequences. It is therefore expected to be possible in principle to
introduce into retroviruses other than lentiviruses the ability to
infect non-dividing or slowly-dividing cells.
[0028] To date, the most widely used retroviral vector systems for
human gene therapy applications have used MLV. However, retroviral
vector systems may also be based on other oncoretroviruses (the
sub-group of retroviruses containing MLV), lentiviruses, or
retroviruses from other sub-groups. Examples of lentiviruses are
HIV SIV, FIV, BLV, EIAV, CAEV and visna virus. Of these, HIV and
SIV are presently best understood. However, preferred for use in
gene therapy would be a non-immunodeficiency lentivirus because the
immunodeficiency viruses inevitably bring with them safety
considerations and prejudices. A range of retroviruses have already
been split into packaging and vector components for retroviral
vector particle production systems, including ASLV, SNV and RSV. It
will be evident that a retroviral vector according to the invention
need not be confined to the components of a particular retrovirus.
The retroviral vector may comprise components derived from two or
more different retroviruses, and may also comprise synthetic
components. Vector components can be manipulated to obtain desired
characteristics, such as target cell specificity.
[0029] The lentivirus group can be split into "primate" and
"non-primate". Examples of primate lentiviruses include the human
immunodeficiency virus (MIV), the causative agent of human acquired
immunodeficiency syndrome (AIDS), and the simian immunodeficiency
virus (SIV). The non-primate lentiviral group includes the
prototype "slow virus" visna/maedi virus (VMV), as well as the
related caprine arhritis-encephalitis virus (CAEV), equine
infectious anaemia virus (ELAV) and the more recently described
feline immunodeficiency virus (FIV) and bovine immunodeficiency
virus (BIV).
[0030] Details on the genomic structure of some lentiviruses may be
found in the art. By way of example, details on HIV and EIAV may be
found from the NCBI Genbank database (i.e. Genome Accession Nos.
AF033819 and AF033820 respectively). Details of HIV variants may
also be found at http://hiv-web.lanl.gov. Details of EIAV variants
may be found through http://www.ncbi.nlm.nih.gov. Further details
on EIAV can be found in U.S. Pat. No. 6,277,633, incorporated
herein by reference.
[0031] Lentiviruses that are the subject of patents and patent
publications and patent applications of Oxford Biomedica are
advantageously employed in the practice of the invention.
[0032] During the process of infection, a retrovirus initially
attaches to a specific cell surface receptor. On entry into the
susceptible host cell, the retroviral RNA genome is then copied to
DNA by the virally encoded reverse transcriptase which is carried
inside the parent virus. This DNA is transported to the host cell
nucleus where it subsequently integrates into the host genome. At
this stage, it is typically referred to as the provirus. The
provirus is stable in the host chromosome during cell division and
is transcribed like other cellular genes. The provirus encodes the
proteins and other factors required to make more virus, which can
leave the cell by a process sometimes called "budding".
[0033] Each retroviral genome comprises genes called gag, pol and
env which code for virion proteins and enzymes. These genes are
flanked at both ends by regions called long terminal repeats
(LTRs). The LTRs are responsible for proviral integration, and
transcription. They also serve as enhancer-promoter sequences. In
other words, the LTRs can control the expression of the viral
genes. Encapsidation of the retroviral RNAs occurs by virtue of a
psi sequence located at the 5' end of the viral genome.
[0034] The LTRs themselves are identical sequences that can be
divided into three elements, which are called U3, R and U5. U3 is
derived from the sequence unique to the 3' end of the RNA. R is
derived from a sequence repeated at both ends of the RNA, and U5 is
derived from the sequence unique to the 5' end of the RNA. The
sizes of the three elements can vary considerably among different
retroviruses.
[0035] For the viral genome, the site of transcription initiation
is at the boundary between U3 and R in the left hand side LTR and
the site of poly (A) addition (termination) is at the boundary
between R and U5 in the right hand side LTR. U3 contains most of
the transcriptional control elements of the provirus, which include
the promoter and multiple enhancer sequences responsive to cellular
and in some cases, viral transcriptional activator proteins. Some
retroviruses have any one or more of the following genes that code
for proteins that are involved in the regulation of gene
expression: tat, rev, tax and rex.
[0036] With regard to the structural genes gag, pol and env
themselves, gag encodes the internal structural protein of the
virus. Gag protein is proteolytically processed into the mature
proteins MA (matrix), CA (capsid) and NC (nucleocapsid). The pol
gene encodes the reverse transcriptase (RT), which contains DNA
polymerase, associated RNase H and integrase (IN), which mediate
replication of the genome. The env gene encodes the surface (SU)
glycoprotein and the transmembrane (TM) protein of the virion,
which form a complex that interacts specifically with cellular
receptor proteins. This interaction leads ultimately to infection
by fusion of the viral membrane with the cell membrane.
[0037] Lentiviruses may also contain "additional" genes which code
for proteins other than gag, pol and env. Examples of additional
genes include in HIV, one or more of vif, vpr, vpx, vpu, tat, rev
and nef. EIAV has, for example, the additional genes S2 and
dUTPase.
[0038] Proteins encoded by additional genes serve various
functions, some of which may be duplicative of a function provided
by a cellular protein. In EIAV, for example, tat acts as a
transcriptional activator of the viral LTR. It binds to a stable,
stem-loop RNA secondary structure referred to as TAR. Rev regulates
and co-ordinates the expression of viral genes through rev-response
elements (RRE). The mechanisms of action of these two proteins are
thought to be broadly similar to the analogous mechanisms in the
primate viruses. The function of S2 is unknown. In addition, an
EIAV protein, Ttm, has been identified that is encoded by the first
exon of tat spliced to the env coding sequence at the start of the
transmembrane protein.
[0039] For the production of retroviral vector particles, the
vector RNA genome is expressed from a DNA construct encoding it, in
a host cell. The components of the particles not encoded by the
vector genome are provided in trans by additional nucleic acid
sequences (the "packaging system", which usually includes either or
both of the gag/pol and env genes) expressed in the host cell. The
set of sequences required for the production of the retroviral
vector particles may be introduced into the host cell by transient
transfection, or they may be integrated into the host cell genome,
or they may be provided in a mixture of ways. The techniques
involved are known to those skilled in the art.
[0040] Certain retroviruses have special characteristics which may
be useful in particular gene therapy applications. For example, the
lentiviruses such as HIV are capable of infecting and transducing
non-dividing and/or slowly-dividing cells because they have means
for getting the proviral DNA across the nuclear membrane of target
cells. This feature will be useful if it is desired to target
non-dividing or slowly-dividing cell types in nucleic acid
transfer. Such cell types include the neurons of the human brain,
which are a potentially important target for gene therapy treatment
of Parkinson's disease. A retroviral vector particle according to
the invention may thus be derived from a lentivirus, at least to
the extent that it is capable of delivering proviral DNA
efficiently to a non-dividing or slowly-dividing cell.
[0041] The vector can comprise a non-lentiviral expression control
element, which will usually be a promoter. This term includes known
promoters, in part or in their entirety, which may be
constitutively acting or may be inducible only under certain
conditions e.g. in the presence of a regulatory protein. This
enables expression of one or more NOI to be restricted e.g. to
particular cell types or to cells in which a particular exogenous
signal is present. For example, heavy metal induction of a NOI
could be achieved by using component of the metallothionein
promoter. Expression control by a steroid hormone may be another
useful approach. Brain specific, stein cell specific or
tumour-specific gene expression signals might alternatively be
used.
[0042] The non-lentiviral promoter replaces the lentiviral
protein-dependent promoter function of the lentiviral 5' LTR. For
HIV, this means that the 5' LTR is no longer responsive to the HIV
Tat protein. Tat acts on the TAR region of R; in an HIV-based
vector according to the invention functional TAR sequences are
therefore absent in order to avoid reductions of translation by the
TAR structure. Enhancer sequences contained in the HIV U3 regions
are also preferably excluded. A straightforward way to achieve the
desired vector LTRs is therefore to replace the lentiviral R
regions and as far as possible the U3 regions, but leaving
essential lentiviral sequences present such as a short sequence of
the U3 region necessary for integration.
[0043] The invention is outlined in FIG. 1. The vector system is
designated Lentiviral LTR-Deleted (LLD) vector. It comprises a DNA
molecule in which a CMV or other high efficiency promoter is used
to drive the expression of the vector RNA in a producer cell. This
strategy is analogous to the ST vector system (Soneoka et al., 1995
Nucl. Acids Res. 23, 628). The producer cell will have been
engineered to produce compatible lentiviral structural proteins and
enzymes. It will be, therefore, what is known as a vector packaging
cell. The producer DNA can be used as an autonomous plasmid that
either does or does not replicate of it can be integrated into the
producer cell genome. All of these strategies are known in the
field (Soneoka et al., 1995 Nucl. Acids Res. 23, 628; Miller and
Rossman 1989 BioTech. 7, 980; Miller 1990 Hum. Gene Ther. 1, 5).
The producer DNA for the vector genome may contain at least the
following contiguous components: a high efficiency promoter, a
non-lentiviral R region that either comes from another retrovirus
or is completely synthetic; all or part of the lentiviral U5 region
that contains sequences required for integration by the lentiviral
integrase and sequences necessary for efficient reverse
transcription; packaging signals that are recognized by the
packaging components of the producer cell; an internal region that
might contain one or more NOI, including therapeutic or reporter
NOI or selectable markers and associated expression signals (in
addition the internal region might contain components of systems
for ensuring efficient RNA splicing and transport); a second:
strand primer site from the lentivirus; a short sequence of 30-100
nucleotides from the lentivirus U3 region that is required for
efficient integration by the lentivirus integrase; a heterologous
promoter that might confer tissue specificity of gene expression or
regulation by an exogenous signal so that a NOI can be expressed
appropriately; and an R region that is identical to the first R
region together with transcription termination and polyadenylation
signals required to produce a vector RNA with terminal R
regions.
[0044] This producer DNA produces an RNA molecule that is packaged
by the lentiviral packaging system. The resulting vector particles
will deliver that RNA to a susceptible cell, the RNA will be
converted to DNA by the lentiviral reverse transcriptase and it
will be integrated into the cells genome by the lentiviral
integrase. The resulting provirus will have the CMV promoter
component of the producer DNA replaced by the short lentiviral
sequence from the end of the lentiviral U3 region and the
heterologous promoter that may confer tissue specific or regulated
gene expression. Because the lentiviral R region has been entirely
replaced, there are no inhibitory TAR sequences in the integrated
vector genome.
[0045] As will be evident, in order to function as a vector, the
lentiviral LTR-deleted vector according to the invention will need
to have a reverse transcription system (compatible reverse
transcriptase and primer binding sites) and an integration system
(compatible integrate and integration sites) allowing conversion to
the provirus and integration of the double-stranded DNA into the
host cell genome. Usually these will include gag and pol proteins
derived from the retrovirus. Additionally, the vector genome will
need to contain a packaging signal. These systems and signals are
described in more detail below in the Examples and will generally
be provided by the retrovirus, on which the vector is based. That
the vector particle according to the invention is "based on" a
retrovirus means that it is derived from that retrovirus. The
genome of the vector particle comprises components from that
retrovirus as a backbone.
[0046] It will be evident also that, although the vector according
to the invention is based on a particular retrovirus, this may be a
genetically or otherwise (e.g. by specific choice of packaging cell
system) altered version of the retrovirus. For example, portions of
the retroviral genome not required for its ability to be packaged,
undergo reverse transcription and integrate, can be excluded. Also,
the vector system can be altered e.g. by using different env genes
to alter the vector host range and cell types infected or
transduced.
[0047] It may be advantageous to include further elements of the
retrovirus on which the vector is based. For HIV this might include
functional rev and RRE sequences, enabling efficient export of
RRE-containing RNA transcripts of the vector genome from the
nucleus to the cytoplasm of the target cell.
[0048] The selected NOI under the control of the exogenous promoter
is or are chosen according to the effect sought to be achieved. For
gene therapy purposes there will be at least one therapeutic NOI
encoding a POI which is active against the condition it is desired
to treat or prevent. Alternatively or additionally, there may be a
selected NOI which acts as a marker by encoding a detectable
product. A NOI may encode, for example, an anti-sense RNA, a
ribozyme, a transdominant negative mutant of a target protein, a
toxin, a conditional toxin, an antigen that induces antibodies or
helper T-cells or cytotoxic T-cells, a single chain antibody or a
tumour suppresser protein.
[0049] Preferably, the retroviral vector according to the invention
is a single transcription unit vector, that is, the vector genome
in DNA or RNA form is under the transcriptional control of no more
than one vector promoter at any one time. In a preferred
embodiment, this is achieved by locating the polynucleotide
sequence according to the invention such that in the DNA form of
the vector genome integrated into the target cell genome (the DNA
provirus), it is under transcriptional control of the 5' LTR. There
are alternative ways of achieving a single transcription unit
vector, however. The vector genome could be designed as a
self-inactivating vector (Yu et al., 1986 PNAS 83, 3194) in which
part of the 3' U3 sequences are deleted so that the transduced
vector genome has a non-functional 5' LTR promoter. The
polynucleotide sequence according to the invention would be
operably linked to an internal conditional promoter between the
LTRs which could be activated once the vector has transduced a
target cell. Activation of the promoter might be dependent upon
cellular or external factors.
[0050] Although single transcription unit vectors are preferred,
other vectors are not excluded. It may be useful for example to
include a marker gene in the vector, operably linked to a different
promoter which may be active simultaneously with the promoter
responsible for transcription of the polynucleotide sequence
encoding the fusion protein. A marker gene encoding a selectable
marker may be useful for selecting successfully transfected
packaging cells, or successfully transduced target cells. Marker
genes encoding selectable markers may be for instance drug
resistance genes or metabolic enzyme genes.
[0051] Where two or more NOI are present and under transcriptional
control of the exogenous promoter, there may be an internal
ribosome entry site (IRES) e.g. from picornaviral RNA, to allow
both NOI to be separately translated from a single transcript.
Retroviruses incorporating IRES sequences have been constructed by
others.
[0052] A further NOI may also be present under the control of a
separate promoter. Such a NOI may encode, for example, a selectable
marker, or a further therapeutic agent which may be among the
therapeutic agents listed herein. Expression of the NOI may be
constitutive; in the case of a selectable marker this may be useful
for selecting successfully transfected packaging cells, or for
packaging cells which are producing particularly high titers of the
retroviral vector particles. Alternatively or additionally, the
selectable marker may be useful for selecting cells which have been
successfully infected with the retroviral vector and have the
provirus integrated into their own genome.
[0053] One way of performing gene therapy is to extract cells from
a patient, infect the extracted cells with a retroviral vector and
reintroduce the cells back into the patient. A selectable marker
may be used to provide a means for enriching for infected or
transduced cells or positively selecting for only those cells which
have been infected or transduced, before reintroducing the cells
into the patient. This procedure may increase the chances of
success of the therapy. Selectable markers may be, for instance,
drug resistance genes, metabolic enzyme genes, or any other
selectable markers known in the art.
[0054] However, it will be evident that for many gene therapy
applications of retroviral vectors, selection for expression of a
marker gene may not be possible or necessary. Indeed expression of
a selection marker, while convenient for in vitro studies, could be
deleterious in vivo because of the inappropriate induction of
cytotoxic T lymphocytes (CTLs) directed against the foreign marker
protein. Also, it is possible that for in vivo applications,
vectors without any internal promoters will be preferable. The
presence of internal promoters can affect, for example, the
transduction titres obtainable from a packaging cell line and the
stability of the integrated vector. Thus, single transcription unit
vectors; which may be bi-cistronic or poly-cistronic, coding for
one or two or more NOI, may be the preferred vector designed for
use in vivo.
[0055] It will be evident that the term "gene" is used loosely
here, and includes any nucleic acid of interest coding for a
desired polypeptide of interest. Usually, the NOI delivered by the
vector according to the invention will be cDNAs.
[0056] The retroviral vector according to the invention may be
constructed according to methods known in the art. It is desirable
that the retroviral vector genome does not encode any unnecessary
polypeptides, that is any polypeptides that are not required for
achieving the effect the vector is designed for. In any case, the
retroviral vector will be replication defective. Particular factors
to be taken into consideration when constructing a retroviral
vector include safety aspects and the avoidance of undesirable
immune responses. Thus, it is necessary to exclude from the vector
genome fill length gag-pol or env coding regions, or preferably
both. Preferably, the retroviral vector genome which will be
inserted into the target cell in the form of a DNA provirus
contains the minimum retroviral material necessary to function.
This avoids both the possible reconstruction of infectious virus
particles, and expression of unwanted virus proteins in the target
cell which could otherwise evoke undesirable immune responses in
the patient being treated.
[0057] The vector according to the invention will also be capable
of infecting and transducing cells which are slowly-dividing, and
which non-lentiviruses such as MLV would not be able to efficiently
infect and transduce. Slowly-dividing cells divide once in about
every three to four days. Mammalian non-dividing and
slowly-dividing cells include brain cells, stem cells, terminally
differentiated macrophages, lung epithelial cells and various other
cell types. Also included are certain tumour cells. Although
tumours contain rapidly dividing cells, some tumour cells
especially those in the centre of the tumour, divide infrequently.
The rate of cell division can easily be determined using
proliferation assays known in the art.
[0058] DNA constructs encoding the vector genome described herein
are preferably linked to a high efficiency promoter such as the CMV
promoter. Other high efficiency promoters are known. This gives
rise to a high level of expression of the vector RNA in the host
cell producing the retroviral vector particles.
[0059] Suitable host or producer cells for use in the invention are
well known in the art. Many retroviruses have already been split
into replication defective genomes and packaging components. For
those which have not the technology is available for doing so. The
producer cell encodes the viral components not encoded by the
vector genome such as the gag, pol and env proteins. The gag, pol
and env genes may be introduced into the producer cell and stably
integrated into the cell genome to give a packaging cell line. The
retroviral vector genome is then introduced into the packaging cell
line by transfection or transduction to create a stable cell line
that has all of the DNA sequences required to produce a retroviral
vector particle. Another approach is to introduce the different DNA
sequences that are required to produce a retroviral vector particle
e.g. the env coding sequence, the gag-pol coding sequence and the
defective retroviral genome into the cell simultaneously by
transient triple transfection (Landau & Littman 1992 J. Virol.
66, 5110; Soneoka et al. 1995).
[0060] The strategy according to the invention has several
advantages in addition to those already described. Firstly, by
making use of a non-lentiviral expression signal for a
transcription unit it is possible to make this vector genome a
single transcription unit genome for both production and expression
in the transduced cell. This avoids the need for internal
promoters. The unpredictable outcome of placing additional
promoters within the retroviral LTR transcription unit is well
documented (Bowtell et al., 1988 J. Virol. 62, 2464; Correll et
al., 1994 Blood 84, 1812; Emerman and Temin 1984 Cell 39, 459;
Ghattas et al., 1991 Mol. Cell. Biol. 11, 5848; Hantzopoulos et
al., 1989 PNAS 86, 3519; Hatzoglou et al., 1991 J. Biol. Chem 266,
8416; Hatzoglou et al., 1988 J. Biol. Chem 263, 17798; Li et al.,
1992 Hum. Gen. Ther. 3, 381; McLachlin et al., 1993 Virol. 195, 1;
Overell et al., 1988 Mol. Cell Biol. 8, 1803; Scharfman et al.,
1991 PNAS 88, 4626; Vile et al., 1994 Gene Ther 1, 307; Xu et al.,
1989 Virol. 171, 331; Yee et al., 1987 PNAS 84, 5197). The factors
involved appear to include the relative position and orientation of
the two promoters, the nature of the promoters and the expressed
nucleic acids and any selection procedures that may be adopted. The
presence of internal promoters can affect both the transduction
titers attainable from a packaging cell line and the stability of
the integrated vector. Loss of gene expression following
transduction can be caused both by provirus deletions and
reversible epigenetic mechanisms of promoter shutdown. In addition,
data from tissue culture studies can often have no bearing on the
performance of the vectors in vivo. These considerations suggest
that simple retroviral vectors containing a single LTR promoter are
likely to be promising vectors for gene therapy (Correll et al.,
1994 Blood 84, 1812). In addition, with the development of
bi-cistronic vectors using only one promoter (Adam et al., 1991 J.
Virol 65, 4985) it will also be possible to produce single
transcription unit vectors coding for two or more NOI, with
correspondingly greater efficacy.
[0061] The second advantage of removing the HIV expression signals
within the U3 and R regions is that these signals are subject to a
number of external influences on their activity. It is known that
the HIV promoter can be activated by a variety of agents such as
UV, stress, other viruses etc. (Peterlin 1992 in Human Retroviruses
ed. Cullen. IRL Press) which makes the transcriptional status of
the vector genome difficult to control. Removal of these sequences
will ensure greater control over the nucleotide to be
expressed.
[0062] In one embodiment, one or more NOI of interest is or are
chosen according to the effect sought to be achieved. The fusion
protein has or is capable of having the desired activity of the
therapeutic gene products. The product encoded by one or more of
the NOI may be an enzyme. The fusion protein may thus display the
activity of one or more enzymes. Where the NOI encode two different
enzymes, the resulting fusion protein is a bifunctional enzyme. In
the specific example described herein, the fusion protein comprises
the enzymes tyrosine hydroxylase and DOPA dehydroxylase having
enzyme activities as described above.
[0063] Preferably the NOI are linked by a sequence encoding a
flexible linker. A suitable linker may comprise amino acid repeats
such as glycine-serine repeats. The purpose of the linker is to
allow the correct formation and/or functioning of the POI. It must
be sufficiently flexible and sufficiently long to achieve that
purpose. Where the NOI encode two different enzymes, the linker
needs to be chosen to allow the functioning of both of the enzymes.
The coding sequence of the flexible linker may be chosen such that
it encourages translational pausing and therefore independent
folding of the protein products of the NOI.
[0064] A person skilled in the art will be able to design suitable
linkers in accordance with the invention. Some specific examples of
suitable linkers are given below; it will be evident that the
invention is not limited to these particular linkers.
[0065] 1. (Gly-Gly-Gly-Gly-Ser)3 as described in Somia et al., 1993
PNAS 90, 7889.
[0066] 2. (Gly-Gly-Gly-Gly-Ser)5, a novel linker.
[0067] 3.
(Asn-Phe-Ile-Arg-Gly-Arg-Glu-Asp-Leu-Leu-Glu-Lys-Ile-Ile-Arg-Gl-
n-Lys-Gly-Ser-Ser-Asn) from HSF-1 of yeast, see Wiederrecht et al.,
1988 Cell 54, 841.
[0068] 4.
(Asn-Leu-Ser-Ser-Asp-Ser-Ser-Leu-Ser-Ser-Pro-Ser-Ala-Leu-Asn-Se-
r-Pro-Gly-Ile-Glu-Gly-Leu-Ser) from POU-specific OCT-1, see Dekker
et al., 1993 Nature 362, 852 and Sturm et al., 1988 Genes and Dev.
2, 1582.
[0069] 5. (Gln-Gly-Ala-Thr-Phe-Ala-Leu-Arg-Gly-Asp-Asn-Pro-GlnGly)
from RGD-containing Laminin peptide, see Aumailly et al., 1990 FEES
Lett. 262, 82.
[0070] 6.
(Ser-Gly-Gly-Gly-Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr-Gly-Gly-Ser-Se-
r-Pro-Gly) from LIDV-containing linker, see Wickham et al., Gene
Therapy 1995 2, 750.
[0071] In addition to gene therapy, the invention has several other
useful applications. The alteration of gene expression, by
upregulating or downregulating the production of gene products can
be accomplished using the vectors of the invention. The vectors of
the invention can also be employed in vitro to produce therapeutic
proteins, to express selectable markers, or for other expression of
assays. Examples of proteins that may be expressed using the
vectors of the invention include, but are not limited to, Factor
VIII, Factor IX, erythropoietin, alpha-1 antitrypsin, calcitonin,
glucocerebrosidase, growth hormone, low density lipoprotein (LDL)
receptor, apolipoproteins (e.g. apolipoprotein E or apolipoprotein
A-I), interleukins, interleukin receptors and antagonists, insulin,
globin, immunoglobulins, catalytic antibodies, superoxide
dismutase, immune responder modifiers, parathyroid hormone,
interferons, growth factors, including insulin-like growth factors
and nerve growth factors, tissue plasminogen activators, colony
stimulating factors, and variants of these proteins.
[0072] In the particular embodiment described herein, the invention
addresses the problems of the prior art by providing a single
fusion gene that expresses a fusion protein composed of TH and DD.
The single gene encodes a single protein with both enzyme
activities. This permits the construction of a simple single
transcription unit retroviral vector that expresses both enzyme
activities efficiently. The fusion gene is designed such that the
enzymes are linked via a flexible linker, the coding sequence of
which has a short cluster of infrequently used codons (Haas et al.,
1996 Curr. Biol. 6, 315) to encourage translational pausing and,
therefore, independent folding of the two domains of the new
bifunctional enzyme. Two different types of fusion gene were made.
In the first the order of the enzyme activities is TH-DD and in the
other it is DD-TH. Both types are made because they may have
different advantages, and properties under different conditions.
Human tyrosine hydroxylase is encoded by a single gene which is
alternatively spliced to create four types of TH that differ
towards their amino terminus. (Grima et al., 1987 Nature 326, 707;
Kaneda et al., 1987 BBRC 146, 971). However, identical primers can
be used to isolate all four cDNAs by PICR as the termini are the
same.
[0073] The following examples are provided as a further description
of the invention, and to illustrate but not limit the
invention.
EXAMPLES
Example 1
An HIV-based LLd Vector with the MLV U3 Promoter and MLV R
Regions
[0074] Lentiviral vectors a are particularly useful for gene
transfer to non-dividing cells. Amongst many important non-dividing
target cells are the neurons of the human brain. These cells might
be target cells for the delivery of thdd or ddth cells for the
treatment of Parkinson's disease. This Example describes the
construction of an HIV based vector which will deliver and express
thdd or ddth genes, for example.
[0075] The structure of a general HIV LLD vector system is shown in
FIG. 2. This example is shown in FIGS. 3 and 4. It is constructed
as follows. the minimal requirements for HIV reverse transcription
are the primer binding site (PBS) to initiate the negative strand
DNA synthesis, the polypurine tract APT) to initiate the positive
DNA synthesis, and identical 5' and 3' R sequences to allow the
first template switch. The incorporation of the PBS and PPT from
HIV-1 into the vector and the R sequences from MLV into both LTRs
is therefore required. As secondary structure within the 5' U5
region might be important for reverse transcription the U5 region
in the 5' LR is from JIV-1. For the U5 region at the 3' LTR, the U5
from HIV-1 was used to make sure correct termination of
transcription occurred at the R-U5 border. However, any termination
signals could be used. For efficient integration, 30 nucleotides at
the 5' end of the HIV-1 U3 at the 3' LTR were incorporated.
[0076] In order for the MLV U3 element to appear in the 5' LTR
after reverse transcription, it must be in the 3' LTR of the viral
RNA. The whole MLV U3 except 30 bps of the 5' end replaced the
HIV-1 U3. The 3' LTR of the vector was designed to contain several
convenient restriction sites, so that the MLV U3 can be easily
replaced by other heterologous promoters (FIG. 4). Any heterologous
promoters will be amplified by PCR with primer containing StuI and
NarI sites at each end and will be used to replace the MLV U3. Not
only StuI but also NheI and Afill may be used at the 5' end of the
promoter cassettes. NarI (GGCGCC) is located on the junction
between the promoter and R, so that the transcription start site
from the heterologous promoter can be preserved. The MLV U3
sequences between XbaI and NarI contains the basic promoter
elements including TATA box, GC box, and CAAT box. Therefore the
MLV enhancer can be replaced by any other enhancers as a StuI (or
NheI or AfIII)-XbaI cassettes.
[0077] For efficient packaging 353 nucleotides of gag is known to
be sufficient (Srinivasakumar et al., 1996 CSH Retrovirus Meeting
abstract). The 353 nucleotides of gag sequences corresponds to the
sequences from 790 to 1144, within this three ATG's (790, 834, 894)
were removed by mutation. In addition a polycloning site is located
downstream of gag.
[0078] In order to achieve efficient export of RNA species encoded
by HIV genome, rev and RRE are required. They are included in the
LLD vector and correspond to sequences 5861 to 6403 and 7621 to
9085 from is HIV-1 (HXB2). Tat coding sequence is not present in
the vector.
[0079] Details of Construction of the Producer DNA:
[0080] A. 5' Structure (All HIV-1 Coordinates are from HXB from the
Los Alamos Sequence Database and MoMLV Sequences are from Shinnick
et al. 1981 Nature 293, 543)
[0081] The 5' half of the vector contains the hybrid 5' LTR (CMV
promoter-MLV R-HIV-1 U5), HIV-1 PBS, and HIV-1 packaging signal.
This will be constructed by recombination PCR. One of the templates
for the PCR, pHIVdge2, is an HIV-1 proviral DNA which has a
mutation created by filling-in and religation at the ClaI site
(831) and a deletion between NdeI (6403) and BgIII (7621). The
junction between MLV R and HIV-1 U5 is created by two primary PCR
reactions (using the primer NIT1 and NIT2; NIT3 and NIT4) and a
secondary PCR reaction (using the primers NIT1 and NIT4). The PCR
product is inserted into pBluesriptKS+ (STRATAGEN) at KpnI and XhoI
site (Construct A1). In order to mutate three ATGs in the gag
region, the primers contain mutated codons. TABLE-US-00001 NIT1:
(SEQ ID NO:1) 5'-ccgggtacccgtattcccaataaagcctcttgctgtttgca-3' NIT2:
(SEQ ID NO:2) 5'-ctacgatctaattctcccccgcttaatactgacgctctcgcacctat
ctc-3' N IT3: (SEQ ID NO:3)
5'-gcgggggagaattagatcgtagggaaaaaattcggttaaggccaggg
ggaaagaaaaaatataaattaaaacatatagtttggg-3' NIT4: (SEQ ID NO:4)
5'-gaattctcgaggcgtgctgtgcttttttctatc-3'
[0082] The CMV promoter--MLV R fragment is amplified by PCR from
pRV109 (Soneoka et al., 1995 Nucl. Acids Res. 23, 628) to contain
KpnI sites at both ends using the PCR primers NIT5 and NIT6 and
inserted into construct A1 to produce construct A2. TABLE-US-00002
NITS: (SEQ ID NO:5) 5'-gtaggtacccgttacataacttacggtaaatg-3' NIT6:
(SEQ ID NO:6) 5'-agaggctttattgggaatacg-3'
[0083] B. 3' Structure
[0084] The 3' half of the vector genome includes the HIV-1 rev
coding region and RRE, PPT, 36 by of 5' end of HIV-1 U3, and the
whole MLV LTR except 30 by of 5' end. The sequences (5861-6000) are
PCR amplified from pHIVdge2 (using NIT7 and NIT8) and are subcloned
into pSP64 (PROMEGA) at BamHI and SacI site (Construct B1).
TABLE-US-00003 (SEQ ID NO:7) NIT7:
5'-cacggatccactagttggaagcatccaggaagtcagc-3' (SEQ ID NO:8) NIT8:
5'-ctctgactgttctgatgagc-3'
[0085] The SacI-SacI fragment (6000-6403 and 7621-9572) from
pHIVdge2 is inserted into the above construct to produce construct
B2. Finally the HIV-1-MLV hybrid LTR will be created by two primary
PCRs (using NIT9 and NIT10 with pHIVdge2 as the template; NIT11 and
NIT12 with pLXSN (Accession number M28248; Miller et al., 1989) as
the template) and one secondary PCR reaction (using NIT9 and
NIT12). The PCR product will be inserted at the XhoI and EcoRI
sites in Construct B2 to produce Construct B3. TABLE-US-00004 NIT9:
(SEQ ID NO:9) 5'-gagcagcatctcgagacctgg-3' NIT10: (SEQ ID NO:10)
5'-tggcgttacttaagctagcaggcctgtcttctttgggagtgtt ttagc-3' NIT11: (SEQ
ID NO:11) 5'-cccaaagaagacaggcctgctagcttaagtaacgccatttttcc-3' NIT12:
(SEQ ID NO:12) 5'-cctgaattccgcggaatgaaagacccccgctgacg-3'
[0086] C. Complete Vector
[0087] The two halves of the vector are combined by inserting the
SpeI-SacII fragment from construct B3 into construct A2. The
resulting construct, C1, possesses a poly-cloning site;
XhoI-SalI-ClaI-HindIII-EcoRV-EcoRI-PstI-SmaI-BamI-SpeI (underlined
sites are unique in the vector). This plasmid is designated pLLD1
and the retroviral vector that it produces is LLD1.
[0088] The .beta.-galactosidase gene was then taken from pSP72-lacZ
(XhoI-BamHI) and inserted into the construct C1 at SaII and BamHI
to produce LLD1-lacZ. This was used to transfect 293T cells
together with plasmids providing the HIV gag and pol components
(pRV664, FIG. 5) and either a plasmid expressing gp160 from HIV
(pRV438 or pSynp160 nm, FIG. 5) or a plasmid expressing the VSVG
protein (pRV67, FIG. 5). Any plasmids encoding the same proteins
would work equally well. The resulting virus that is produced
transduced the lacZ gene to CD4+ Hela cells in the case of virus
containing gp160 and to CD4-Hela cells in the case of the VSVG
bearing virus. In addition the VSVG bearing virus delivers lacZ to
post-mitotic neurones. In each case the expression of the lacZ gene
is high, as determined by Xgal staining, and independent of
Tat.
[0089] Alternatively, ddth1 is used to illustrate the principle of
a fusion construct, but any of the fusion genes could be used.
Plasmid pX1 is cut with HincII and SpeI and the fragment purified.
This is then inserted into LLD1 cut with EcoRV and SpeI to create
pLLD1:thdd1. When this is transfected into a packaging cell line
(suitable packaging components are shown in FIG. 5) and viral
vector particles produced, those vector particles deliver the thdd
gene to the recipient cells where the fusion enzymes are expressed.
Such a retroviral vector system is useful for the treatment of
Parkinson's disease by gene therapy.
Example 2
Other LLD Vectors
[0090] Systems similar to that described in Example 1 can be
produced from other lentiviruses. These systems avoid using HIV,
with its associated perceived risks as a gene delivery system. For
example constructions could be designed using sequence information
from FIV (Talbott et al., 1989 PNAS 86, 5743), EIAV (Payne et al.,
1994 J. Gen. Virol. 75, 425), Visna-virus (Sonigo et al., 1985 Cell
42, 369; Querat et al., 1990 Virology 175, 434), BIV (Garvey et
al., 1990 Virology 175, 391), CAEV (Saltarelli et al., 1990
Virology 179, 347) and SIV (Los Alamos sequence database).
Example 3
Construction of TH-DD Fusion Genes Designated thdd1-4 (FIG. 7)
[0091] A human brain Substantia nigra cDNA library (Clontech.
HI-3009a & b) is used as template DNA in a PCR amplification of
the TH and DD cDNAs. The primers are shown in FIG. 9. In the case
of the four TH cDNAs representing the HTH-1 to HTH-4 genes (Grima
et al.; Kaneda et al.), they are all treated in the same way from a
pool of PCR products and then identified after cloning and
sequencing. The TH PCR products are produced from linkers
containing a HincII site at the 5' end of the nucleic acid and a
flexible linker and HindIII site at the 3' end. The flexible linker
amino acid sequence is (Gly.sub.4-Ser).sub.3, a sequence often used
to link the two chains of an antibody to produce an scFv (e.g.
Somia et al., 1995 PNAS 92, 7570). The human DD PCR product was
designed to have a HindIII site at the 5' end and a SpeI site at
the 3' end. The two fragment are ligated and the ligated products
of the correct size (2.98 kb, 2.99 kb, 3.06 kb and 3.07 kb for the
four variants) are purified from an agarose gel. The purified
fragments are then inserted into pBLUEscriptKS+ using HincII and
SpeI. Thins ligation mixture is used to transform E. coli (XL2-Blue
ex. Stratagene 200249) and clones were used to prepare DNA which is
then sequenced to ensure that the genes are intact and to identify
HTH1-4. Plasmids containing fragments encoding the four different
HTH coding sequences fused to DD are designated pthdd1-4. The the
HincII-SpeI fragments from these plasmids are then inserted into
the mammalian ex pression vector pCl-neo (Promega E1841). This is
achieved by cutting pCl-neo with XhoI and SmaI and cutting the
pBluescriptKS+ derived plasmids with SpeI and blunt ending and then
cutting with XhoI. The cut products are then ligated together and
correct plasmids checked by milipreps. The pCl-neo plasmids
containing the fusion genes are designated pClthdd1-4. These are
then used to transiently transfect 293T cells which rare then
assayed for TH and DD by the methods of Waymire et al. (1971)
(Anal. Biochem. 43, 588) and the method described in Anal. Biochem.
(1984139, 73). In each case significantly increased levels of TH
and DD are seen compared with control cells transfected only with
pCl-neo. This demonstrates that the fusion genes expresses fusion
proteins with both activities.
Example 4
Construction of DD-NTI Fusion Genes Designated ddth1-4 (FIG. 8)
[0092] The construction of these genes is identical to that of
Example 3 but the DD and TH coding sequences are in reciprocal
locations. Similarly dual enzyme activities are encoded by the
ddth1-4 genes.
Example 5
Construction of a Retroviral Vector Expressing a thdd Gene is a
Single Transcription Unit Configuration
[0093] The thdd and ddth genes are useful for the gene therapy of
Parkinson's disease. They can be used in a wide range of vectors
but they are particularly suited to single transcription unit
retroviral vectors. An example of such a vector is produced as
follows: Starting with pLNSX (Miller) a polylinker is inserted into
the vector. Briefly, a SspI/HindIII fragment, containing the
polylinker from pBluescriptKS+ is inserted into pLNSX cut with SspI
and HindIII. The resulting plasmid is known as pMLD1. Plasmid, pX1,
for example, is then cut with SpeI and then the ends filled in with
DNA polymerase. The plasmid is then cut again with XhoI. The
resulting thdd fragment is then inserted into pMLD1 cut with XhoI
and ClaI (blunt-ended) to produce the resulting molecule
pMLD1:thdd1. When this plasmid is used to transfect a packaging
cell line retroviral vectors are produced which transduce
susceptible cells with the thdd gene in a single transcription unit
configuration. In this case the gene is expressed from the MLV LTR
promoter but any promoter inserted into the 5' LTR via a U3
replacement or similar strategy would be equally effective.
[0094] Having thus described in detail preferred embodiments of the
present invention, it is to be understood that the invention
defined by the appended claims is not to be limited to particular
details set forth in the above description, as many apparent
variations thereof are possible without departing from the spirit
or scope of the present invention. Modifications and variations of
the method and apparatuses described herein will be obvious to
those skilled in the art, and are intended to be encompassed by the
following claims.
Sequence CWU 1
1
28 1 41 DNA Unknown Primer NIT1 used in PCR reaction to create
junction between MLV R and HIV-1 U5 1 ccgggtaccc gtattcccaa
taaagcctct tgctgtttgc a 41 2 50 DNA Unknown Primer NIT2 used in PCR
reaction to create junction between MLV R and HIV-1 U5 2 ctacgatcta
attctccccc gcttaatact gacgctctcg cacctatctc 50 3 84 DNA Unknown
Primer NIT3 used in PCR reaction to create junction between MLV R
and HIV-1 U5 3 gcgggggaga attagatcgt agggaaaaaa ttcggttaag
gccaggggga aagaaaaaat 60 ataaattaaa acatatagtt tggg 84 4 33 DNA
Unknown Primer NIT4 used in PCR reaction to create junction between
MLV R and HIV-1 U5 4 gaattctcga ggcgtgctgt gcttttttct atc 33 5 32
DNA Unknown PCR primer NIT5 used in amplification of CMV promoter 5
gtaggtaccc gttacataac ttacggtaaa tg 32 6 21 DNA Unknown PCR primer
NIT6 used in amplification of CMV promoter 6 agaggcttta ttgggaatac
g 21 7 37 DNA Unknown PCR primer NIT7 used in amplification of
pHIVdge2 7 cacggatcca ctagttggaa gcatccagga agtcagc 37 8 20 DNA
Unknown PCR primer NIT8 used in amplification of pHIVdge2 8
ctctgactgt tctgatgagc 20 9 21 DNA Unknown PCR primer NIT9 used to
prepare HIV-1-MLV hybrid LTR 9 gagcagcatc tcgagacctg g 21 10 48 DNA
Unknown PCR primer NIT10 used to prepare HIV-1-MLV hybrid LTR 10
tggcgttact taagctagca ggcctgtctt ctttgggagt gttttagc 48 11 44 DNA
Unknown PCR primer NIT11 used to prepare HIV-1-MLV hybrid LTR 11
cccaaagaag acaggcctgc tagcttaagt aacgccattt ttcc 44 12 35 DNA
Unknown PCR primer NIT12 used to prepare HIV-1-MLV hybrid LTR 12
cctgaattcc gcggaatgaa agacccccgc tgacg 35 13 33 DNA Unknown TH5-1
mammalian primer used to amplify the tyrosine hydroxyase (TH) gene
from cDNA library 13 cacagtcgac catgcccacc cccgacgcca cca 33 14 77
DNA Unknown TH3-1 mammalian primer used to amplify the tyrosine
hydroxyase (TH) gene from cDNA library 14 cgtacaagct tcgatcctcc
acctcccgag ccacctccgc ctgaaccgcc tccaccgcca 60 atggcactca gcgcatg
77 15 32 DNA Unknown DD5-1 mammalian primer used to amplify the
DOPA decarboxylase (DD) gene from cDNA library 15 acgcaaagct
tatgaacgca agtgaattcc ga 32 16 34 DNA Unknown DD3-1 mammalian
primer used to amplify the DOPA decarboxylase (DD) gene from cDNA
library 16 ctggactagt ctactccctc tctgctcgca gcac 34 17 32 DNA
Unknown DD5-2 mammalian primer used to amplify the DOPA
decarboxylase (DD) gene from cDNA library 17 cacagtcgac catgaacgca
agtgaattcc ga 32 18 77 DNA Unknown DD3-2 mammalian primer used to
amplify the DOPA decarboxylase (DD) gene from cDNA library 18
cgtacaagct tcgatcctcc acctcccgag ccacctccgc ctgaaccgcc tccaccctcc
60 ctctctgctc gcagcac 77 19 33 DNA Unknown TH5-2 mammalian primer
used to amplify the tyrosine hydroxyase (TH) gene from cDNA library
19 acgcaaagct tatgcccacc cccgacgcca cca 33 20 34 DNA Unknown TH3-2
mammalian primer used to amplify the tyrosine hydroxyase (TH) gene
from cDNA library 20 ctggactagt ctagccaatg gcactcagcg catg 34 21 15
PRT Artificial Sequence flexible linker sequence used to link
fusion proteins 21 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser 1 5 10 15 22 25 PRT Artificial Sequence flexible linker
sequence used to link fusion proteins 22 Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser
Gly Gly Gly Gly Ser 20 25 23 26 PRT Saccharomyces cerevisiae 23 Asn
Phe Ile Arg Gly Arg Glu Asp Leu Leu Gly Lys Ile Ile Arg Gln 1 5 10
15 Lys Ile Ile Arg Gln Lys Gly Ser Ser Asn 20 25 24 23 PRT
Artificial Sequence flexible linker sequence used to link fusion
proteins 24 Asn Leu Ser Ser Asp Ser Ser Leu Ser Ser Pro Ser Ala Leu
Asn Ser 1 5 10 15 Pro Gly Ile Glu Gly Leu Ser 20 25 14 PRT
Artificial Sequence flexible linker sequence used to link fusion
proteins 25 Gln Gly Ala Thr Phe Ala Leu Arg Gly Asp Asn Pro Gln Gly
1 5 10 26 18 PRT Artificial Sequence flexible linker sequence used
to link fusion proteins 26 Ser Gly Gly Gly Glu Ile Leu Asp Val Pro
Ser Thr Gly Gly Ser Ser 1 5 10 15 Pro Gly 27 43 DNA Human
immunodeficiency virus 27 gctaattcac tcccaaagaa gacaggcctg
ctagcttaag taa 43 28 17 DNA Human immunodeficiency virus 28
tcactcgggg cgccagt 17
* * * * *
References