U.S. patent application number 13/117451 was filed with the patent office on 2011-12-01 for method for vector delivery.
This patent application is currently assigned to Oxford BioMedica (UK) Ltd.. Invention is credited to Kyriacos A. Mitrophanous, Scott Ralph, Peter WIDDOWSON.
Application Number | 20110293571 13/117451 |
Document ID | / |
Family ID | 45003388 |
Filed Date | 2011-12-01 |
United States Patent
Application |
20110293571 |
Kind Code |
A1 |
WIDDOWSON; Peter ; et
al. |
December 1, 2011 |
METHOD FOR VECTOR DELIVERY
Abstract
Provided is a lentiviral vector for delivery to the brain for
use in treating a neurological condition, wherein the lentiviral
vector is delivered directly to the brain by delivering the
lentiviral vector via six or fewer tracts per hemisphere, at a
single deposit point per tract.
Inventors: |
WIDDOWSON; Peter; (US)
; Ralph; Scott; (US) ; Mitrophanous; Kyriacos
A.; (US) |
Assignee: |
Oxford BioMedica (UK) Ltd.
Oxford
GB
|
Family ID: |
45003388 |
Appl. No.: |
13/117451 |
Filed: |
May 27, 2011 |
Current U.S.
Class: |
424/93.6 ;
435/320.1 |
Current CPC
Class: |
A61K 31/711 20130101;
A61P 25/08 20180101; A61P 25/00 20180101; C12N 15/86 20130101; A61P
25/16 20180101; A61P 25/18 20180101; A61P 25/28 20180101; A61M
5/178 20130101; A61K 48/005 20130101; C12N 2740/15043 20130101;
A61P 25/06 20180101; A61P 25/04 20180101; A61P 25/14 20180101; A61K
48/0083 20130101; A61K 48/0075 20130101 |
Class at
Publication: |
424/93.6 ;
435/320.1 |
International
Class: |
A61K 35/76 20060101
A61K035/76; A61P 25/00 20060101 A61P025/00; C12N 15/63 20060101
C12N015/63 |
Foreign Application Data
Date |
Code |
Application Number |
May 28, 2010 |
GB |
1009052.0 |
Jan 12, 2011 |
GB |
1100502.2 |
Apr 28, 2011 |
GB |
1107184.2 |
Claims
1. A lentiviral vector for delivery to the brain for use in
treating a neurological condition, wherein a composition comprising
the lentiviral vector is delivered directly to the brain by
continuous infusion using a cannula and wherein between 10-600
.mu.L of the vector composition is delivered per tract at a flow
rate of at least 2 .mu.L/min.
2. The lentiviral vector according to claim 1, wherein the cannula
is of sufficiently narrow bore to prevent substantial backflow of
the vector composition.
3. The lentiviral vector according to claim 1, wherein a constant
flow rate is maintained during infusion of the lentiviral
vector.
4. The lentiviral vector according to claim 1, wherein the flow
rate is increased during infusion of the lentiviral vector.
5. The lentiviral vector according to claim 1, which is an equine
infectious anaemia virus (EIAV) vector.
6. The EIAV vector according to claim 5, which comprises nucleotide
sequences encoding Tyrosine Hydroxylase, GTP-cyclohydrolase I and
Aromatic Amino Acid Dopa Decarboxylase.
7. The lentiviral vector according to claim 1, wherein the
lentiviral vector is delivered via a single tract per
hemisphere.
8. The lentiviral vector according to claim 7, wherein the infusion
has a volume of about 50 .mu.L.
9. The lentiviral vector according to claim 1, wherein the flow
rate at which the vector is delivered is between 2-4 .mu.L/min.
10. The lentiviral vector according to claim 1, wherein the flow
rate at which the vector is delivered is about 3 .mu.L/min.
11. The lentiviral vector according to claim 1, wherein the
lentiviral vector is delivered using a cannula with a bore
equivalent to or narrower than 28 gauge.
12. The lentiviral vector according to claim 1, for treating
Parkinson's disease.
13. A method for treating a neurological disorder in a subject
which comprises the step of administrating a lentiviral vector as
defined in any preceding claim to the subject, in which method a
composition comprising the lentiviral vector is delivered directly
to the brain by continuous infusion using a cannula and wherein
between 10-600 .mu.L of the vector composition is delivered per
tract at a flow rate of at least 2 .mu.L/min.
14. A method for improving the distribution volume of a lentiviral
vector in the putamen when administered directly to the brain of a
subject, wherein the method comprises continuous infusion of a
composition comprising a vector using a cannula, wherein between
10-600 .mu.L of the vector composition is delivered per tract at a
flow rate of at least 2 .mu.L/min.
15. A kit for delivering the lentiviral vector as defined in claim
1 directly to the brain of the subject, which comprises one or more
cannulas.
16. The kit according to claim 15, wherein the cannula(s) is/are
pre-filled with the lentiviral vector at a volume of between 10 and
600 .mu.L.
17. The kit according to claim 15, which comprises one or more
cannula(s) for delivery of the vector, wherein the cannula(s)
is/are 28 gauge or narrower.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to Great
Britain applications 1009052.0, filed on May 28, 2010; 1100502.2,
filed on Jan. 12, 2011; and 1107184.2, filed on Apr. 28, 2011. The
foregoing applications are hereby incorporated by reference in
their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to a lentiviral vector for
delivery to the brain for use in treating a neurological condition.
The lentiviral vector is delivered directly to the brain by
continuous infusion using a narrow bore delivery device with a
reduced number of deposit points compared to previous methods.
BACKGROUND TO THE INVENTION
[0003] Virus-based approaches are known to treat various
neurological diseases, through the introduction of therapeutic
genes to transduce neuronal and/or support cells. For example, a
multicistronic lentiviral vector product, ProSavin.RTM., has been
developed to treat Parkinson's disease. ProSavin.RTM. mediates
intrastriatal dopamine production by transduction into non-dopamine
cells the genes for aromatic L-amino acid decarboxylase, tyrosine
hydroxylase, and GTP cyclohydrolase I (Azzouz et at (2002) J
Neurosci.22: 10302-10312).
[0004] Previous methods of lentiviral vector delivery have
introduced the vectors to specific regions within the brain through
multiple small volume deposits at a low discontinuous flow rate to
ensure sufficient transduction of target cells over a wide area
(Azzouz et at (2002) J Neurosci 22: 10302-10312). For example,
ProSavin.RTM. is administered using multiple tracts (up to 5 per
hemisphere) using a step-wise delivery method which involves
multiple deposits of the vector along each tract (Jarraya et at
(2009) Sci Transl Med 14: 1(2) 2-4).
[0005] Such an approach requires complex pre-surgical planning to
determine the positioning of the cannula tracts and time-consuming
surgery, with an increased risk of bleeding and other surgical
complications associated with the use of multiple cannula tracts to
introduce the vector.
[0006] There is thus a need for improved delivery methods for such
lentiviral vectors.
[0007] There are reports of using convection-enhanced delivery
(CED) as an efficient method of delivering therapeutic agents,
including maghemite nanoparticles, liposomes and small viral
vectors, such as adeno-associated virus vectors (AAV), into the
brain (Lieberman et at (1985) J. Neurosurg. 82:1021-1029;
Bankiewicz et at (2000) Exp. Neurol. 164:2-14; Cunningham et al
(2000) Cell Transplant 9:585-594; Nguyen et at (2001) Neuroreport
12:1961-1964; Mamot et al (2004) J Neurooncol. 68:1-9; Hadaczek et
at (2006) Hum. Gene Ther 17:291-302 and Perlstein et at (2008)
Neuro-Oncol 10:153-161). Using a pressurised infusate, the
distribution of particles and macromolecules through the
perivascular space has been reported to be enhanced above that
achieved by diffusion alone (Chen et at (2004) J. Neurosurg.
101:314-322 and Hadaczek et al (2009) Hum. Gene Ther
20:229-237).
[0008] CED uses a pressure gradient established at the tip of an
infusion catheter that initially creates bulk flow that "pushes"
the therapeutic agent through the space between brain cells.
[0009] Although there are reports of successful use of CED to
deliver small AAVs (Bankiewicz et at (2000) Exp. Neurol. 164:2-14;
Cunningham et at (2000) Cell Transplant 9:585-594; and Hadaczek et
at (2009) Hum. Gene Ther 20:229-237) these findings have little
impact on the delivery of lentiviral vectors, because the
intracellular space in the brain has been calculated as being
between 38 and 64 nm (Thorne and Nicholson (2006) PNAS 104;
5567-5572), whereas the typical diameter of lentiviruses is around
100 nm, typically around four-fold larger than AAV vectors (Fields
Virology Fifth Edition (2007) Eds. Knipe and Howley. Lippincott
Williams and Wilkins). AAV vectors are non-enveloped viruses with a
diameter of around 18-26 nm and are considerably smaller than the
calculated intracellular space.
[0010] Consideration to the cellular tropism (Davidson et at (2000)
Proc. Natl. Acad. Sci. USA, Vol. 97, PP. 3428-3432; Azzouz et at
(2002) J Neurosci 22:10302-10312 and Eschemacher et al (2004) Exp
Med 90:61-69) is an important factor when changing vector delivery,
as neuronal or other cellular targets may be significantly
compromised when vectors are delivered in an accelerated fashion.
Moreover, the relative immunological response to central vector
administration may also be altered when modifying the methodology
for surgical administration into specific brain regions.
DESCRIPTION OF THE FIGURES
[0011] FIGS. 1A-1C show staining for anti-.beta.-galactosidase in
non-human primate putamen following administration of 50 .mu.L
EIAV-LacZ vector suspension using (A) 5 needle tracts, each
delivering 10 .mu.L at 3 points with a 23-gauge Hamilton stainless
steel needle (B) a single infusion at 3 .mu.L/min through a
28-gauge fused silica cannula or (C) infusion of 50 .mu.L of TSSM
formulation buffer only through a 28-gauge fused silica cannula at
1 .mu.L/min. This figure illustrates the superior medio-lateral and
dorso-ventral spread of EIAV vector in the putamen following
administration using a single infusion (B) compared to the
established method of 5 needle tracts (A) in which vector is more
confined to the proximity of the injection tract. The high power
images indicate the neuronal morphology of the EIAV transduced
cells which is similar with both delivery methods. The TSSM buffer
injection provides a negative control for the histological
staining.
[0012] FIG. 2 shows estimation of the volume of vector distribution
in non-human primate putamen using stereology methods following
administration of 50 .mu.L EIAV-LacZ vector suspension using
different delivery methods.
[0013] The data illustrate that administration of vector using a
single infusion through a 28-gauge fused silica cannula at a
constant flow rate of either 1 or 3 .mu.L/min mediates improved
distribution of vector in the putamen compared to the 5 tract
delivery method using a 23-gauge needle and syringe. The higher
flow rate of 3 .mu.L/min demonstrated a greater volume of vector
distribution with the single infusion than the slower rate. Vector
delivery using a single infusion with the 23-gauge needle and
syringe resulted in a lower volume of vector distribution than both
of the methods described above indicating that the gauge of needle
is critical for achieving an improved vector distribution in the
brain.
[0014] FIGS. 3A-3B show low power photomicrographs showing
CD68-positive staining of activated microglia in sections which
received 50 .mu.L EIAV-LacZ vector suspension using (A) 5 needle
tracts with a 23-gauge Hamilton stainless steel needle or (B) a
single infusion through a 28-gauge fused silica cannula. This
figure illustrates that for both delivery methods the inflammatory
response is local and confined to the area of the needle tract.
SUMMARY OF ASPECTS OF THE INVENTION
[0015] The present inventors have surprisingly found that despite
the size of lentiviral vectors relative to the extracellular space,
it is possible to modify the multiple-tract discontinuous delivery
method described for ProSavin.RTM., increase the volume delivered
per tract and the flow rate of infusion for lentiviral vectors
which in turn results in a greater volume of vector spread within
the brain.
[0016] Using a lentiviral vector based on the equine infectious
anaemia virus (EIAV), expressing the reporter gene
.beta.-galactosidase (EIAV-LacZ), they have shown that a single
continuous infusion of this genetically modified lentiviral vector
distributes effectively within the putamen of cynomolgus macaques.
Although vector spread in the rostro-caudal axis of the putamen was
marginally less than using the previously described multiple 5
needle tract approach to manually spread out the vector
distribution, vector distribution in the medio-lateral and
dorso-ventral axes with the continuous single infusion paradigm was
better than the 5-tract multiple deposit approach. Moreover the
total volume of vector distribution in the brain was almost 2-fold
greater with a single continuous infusion compared with the
five-tract multiple deposit method.
[0017] Despite the increased volume and increased flow rate of
administration, the continuous infusion system produced no overt
neuronal damage in the region of vector spread and no evidence of
damage to the blood-brain barrier. Animals did not display any
signs of major toxicity or overt inflammatory responses and no
abnormal clinical signs or motor disturbances were observed. This
is surprising given the relatively large size of lentiviral
vectors. In addition there was less evidence of backflow along the
outer surface of the infusion cannula, which had previously been
observed with the 23-gauge needle using the 5-tract approach.
[0018] This shows that, contrary to expectations in view of their
large size, rather than requiring a slow, discontinuous (multiple
deposit) infusion of small volumes, large volumes of lentiviral
vectors can be "pushed" through the neuronal matrix between cells
using fluid convection without causing obvious signs of tissue
damage and resulting in superior vector distribution within the
target area.
[0019] This results in a reduction in the number of tracts required
to deliver a given volume of infusate. Thus the increased flow-rate
and increased volumes that can be delivered reduces both surgery
time and the risks associated with placement of many cannula sites,
as well as allowing the delivery of higher doses of lentiviral
vector.
[0020] It was also found that a narrow gauge cannula resulted in a
better volume of vector distribution than a wide bore cannula. It
is thought that this is due to reduced back-flow with the narrow
gauge cannula and an increase in pressure from the more narrow
cannula enhancing the vector distribution. Backflow may be a key
problem when delivering a therapeutic under pressure, because if
backflow occurs, a significant amount of vector could be lost up
the cannula tract and would not be available for delivery to the
target area.
[0021] Thus, in a first aspect, the present invention provides a
lentiviral vector for delivery to the brain for use in treating a
neurological condition, wherein a composition comprising the
lentiviral vector is delivered directly to the brain by continuous
infusion using a cannula and wherein between 10-600 .mu.L of the
vector composition is delivered per tract at a flow rate of at
least 2 .mu.L/min.
[0022] The cannula may be of sufficiently narrow bore to prevent
substantial backflow of the vector composition.
[0023] The flow rate may be constant or increasing during infusion
of the lentiviral vector.
[0024] The vector may be an equine infectious anaemia virus (EIAV)
vector, for example an EIAV vector which comprises nucleotide
sequences encoding Tyrosine Hydroxylase, GTP-cyclohydrolase I and
Aromatic Amino Acid Dopa Decarboxylase.
[0025] The lentiviral vector may be delivered via a single cannula
tract per hemisphere.
[0026] The infusion may have a volume of about 50 .mu.L.
[0027] The flow rate at which the vector is delivered may be
between 2-6 .mu.L/min, for example about 3 .mu.L/min.
[0028] The lentiviral vector may be delivered using a cannula with
a bore equivalent to or narrower than 28 gauge.
[0029] The lentiviral vector may be for treating Parkinson's
disease.
[0030] In a second aspect, the present invention provides a method
for treating a neurological disorder in a subject which comprises
the step of administrating a lentiviral vector as defined in any
preceding claim to the subject, in which method a composition
comprising the lentiviral vector is delivered directly to the brain
by continuous infusion using a cannula and wherein between 10-600
.mu.L of the vector composition is delivered per tract at a flow
rate of at least 2 .mu.L/min.
[0031] In a third aspect there is provided a method for improving
the distribution volume of a lentiviral vector in the putamen when
administered directly to the brain of a subject, by continuous
infusion using a cannula, wherein between 10-600 .mu.L of the
vector composition is delivered per tract at a flow rate of at
least 2 .mu.L/min.
[0032] In a fourth aspect there is provided a kit for delivering a
lentiviral vector according to the first aspect of the invention
directly to the brain of the subject, which comprises one or more
cannulas.
[0033] The cannulas may be pre-filled with the lentiviral vector
composition at a volume of between 10 and 600 .mu.L.
[0034] The kit may comprise one or more cannulas for delivery of
the vector, wherein the cannula(s) is/are 28 gauge or narrower.
DETAILED DESCRIPTION
[0035] The present invention relates to a lentiviral vector for
delivery to the brain.
Lentiviral Vectors
[0036] The lentiviral vector according to the present invention may
be derived from or may be derivable from any suitable lentivirus. A
recombinant lentiviral particle is capable of transducing a target
cell with a nucleotide of interest (NOI). Once within the cell the
RNA genome from the vector particle is reverse transcribed into DNA
and integrated into the genome of the target cell.
[0037] Lentiviral vectors are part of a larger group of retroviral
vectors. A detailed list of lentiviruses may be found in Coffin et
al. (1997) "Retroviruses" Cold Spring Harbor Laboratory Press Eds:
J M Coffin, S M Hughes, H E Varmus pp 758-763). In brief,
lentiviruses can be divided into primate and non-primate groups.
Examples of primate lentiviruses include but are not limited to:
the human immunodeficiency virus (HIV), the causative agent of
human auto-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 arthritis-encephalitis virus (CAEV),
equine infectious anaemia virus (EIAV) and the more recently
described feline immunodeficiency virus (FIV) and bovine
immunodeficiency virus (BIV).
[0038] Lentiviruses differ from other members of the retrovirus
family in that lentiviruses have the capability to infect both
dividing and non-dividing cells (Lewis et at (1992) EMBO J
11(8):3053-3058) and Lewis and Emerman (1994) J Virol 68
(1):510-516). In contrast, other retroviruses--such as MLV--are
unable to infect non-dividing or slowly dividing cells such as
those that make up, for example, muscle, brain, lung and liver
tissue.
[0039] A lentiviral vector, as used herein, is a vector which
comprises at least one component part derivable from a lentivirus.
Preferably, that component part is involved in the biological
mechanisms by which the vector infects cells, expresses genes or is
replicated.
[0040] The basic structure of retrovirus and lentivirus genomes
share many common features such as a 5' LTR and a 3' LTR, between
or within which are located a packaging signal to enable the genome
to be packaged, a primer binding site, integration sites to enable
integration into a host cell genome and gag, pol and env genes
encoding the packaging components--these are polypeptides required
for the assembly of viral particles. Lentiviruses have additional
features, such as rev and RRE sequences in HIV, which enable the
efficient export of RNA transcripts of the integrated provirus from
the nucleus to the cytoplasm of an infected target cell.
[0041] In the provirus, the viral genes are flanked at both ends by
regions called long terminal repeats (LTRs). The LTRs are
responsible for proviral integration, and transcription. LTRs also
serve as enhancer-promoter sequences and can control the expression
of the viral genes.
[0042] 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
viruses.
[0043] In a defective lentiviral vector genome gag, pol and env may
be absent or not functional. The R regions at both ends of the RNA
are repeated sequences. U5 and U3 represent unique sequences at the
5' and 3' ends of the RNA genome respectively.
[0044] In a typical lentiviral vector of the present invention, at
least part of one or more protein coding regions essential for
replication may be removed from the virus. This makes the viral
vector replication-defective. Portions of the viral genome may also
be replaced by an NOI in order to generate a vector comprising an
NOI which is capable of transducing a target non-dividing host cell
and/or integrating its genome into a host genome.
[0045] In one embodiment the lentiviral vectors are non-integrating
vectors as described in WO 2007/071994.
[0046] In a further embodiment the vectors have the ability to
deliver a sequence which is devoid of or lacking viral RNA. In a
further embodiment a heterologous binding domain (heterologous to
gag) located on the RNA to be delivered and a cognate binding
domain on gag or pol can be used to ensure packaging of the RNA to
be delivered. Both of these vectors are described in WO
2007/072056.
[0047] The lentiviral vector may be a "non-primate" vector, i.e.,
derived from a virus which does not primarily infect primates,
especially humans.
[0048] The examples of non-primate lentivirus may be any member of
the family of lentiviridae which does not naturally infect a
primate and may include a feline immunodeficiency virus (FIV), a
bovine immunodeficiency virus (BIV), a caprine arthritis
encephalitis virus (CAEV), a Maedi visna virus (MVV) or an equine
infectious anaemia virus (EIAV).
[0049] In a particularly preferred embodiment the viral vector is
derived from EIAV. EIAV has the simplest genomic structure of the
lentiviruses and is particularly preferred for use in the present
invention. In addition to the gag, pol and env genes EIAV encodes
three other genes: tat, rev, and S2. Tat acts as a transcriptional
activator of the viral LTR (Derse and Newbold (1993) Virology
194(2):530-536 and Maury et at (1994) Virology 200(2):632-642) and
Rev regulates and coordinates the expression of viral genes through
rev-response elements (RRE) (Martarano et al. (1994) J Virol
68(5):3102-3111). The mechanisms of action of these two proteins
are thought to be broadly similar to the analogous mechanisms in
the primate viruses (Martarano et al. (1994) J Virol
68(5):3102-3111). 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.
[0050] Preferred vectors of the present invention are recombinant
lentiviral vectors.
[0051] The term "recombinant lentiviral vector" refers to a vector
with sufficient lentiviral genetic information to allow packaging
of an RNA genome, in the presence of packaging components, into a
viral particle capable of infecting a target cell. Infection of the
target cell may include reverse transcription and integration into
the target cell genome. The recombinant lentiviral vector carries
non-viral coding sequences which are to be delivered by the vector
to the target cell. A recombinant lentiviral vector is incapable of
independent replication to produce infectious lentiviral particles
within the final target cell. Usually the recombinant lentiviral
vector lacks a functional gag-pol and/or env gene and/or other
genes essential for replication. The vector of the present
invention may be configured as a split-intron vector. A split
intron vector is described in PCT patent application WO
99/15683.
[0052] Preferably the recombinant lentiviral vector of the present
invention has a minimal viral genome.
[0053] As used herein, the term "minimal viral genome" means that
the viral vector has been manipulated so as to remove the
non-essential elements and to retain the essential elements in
order to provide the required functionality to infect, transduce
and deliver a nucleotide sequence of interest to a target host
cell. Further details of this strategy can be found in our WO
98/17815.
[0054] In one embodiment of the present invention, the vector is a
self-inactivating vector.
[0055] By way of example, self-inactivating retroviral vectors have
been constructed by deleting the transcriptional enhancers or the
enhancers and promoter in the U3 region of the 3' LTR. After a
round of vector reverse transcription and integration, these
changes are copied into both the 5' and the 3' LTRs producing a
transcriptionally inactive provirus (Yu et at (1986) Proc. Natl.
Acad. Sci. 83:3194-3198; Dougherty and Temin et at (1987) Proc.
Natl. Acad. Sci. 84:1197-1201; Hawley (1987) Proc. Natl. Acad. Sci.
84:2406-2410 and Yee et at (1987) Proc. Natl. Acad. Sci.
91:9564-9568). However, any promoter(s) internal to the LTRs in
such vectors will still be transcriptionally active. This strategy
has been employed to eliminate effects of the enhancers and
promoters in the viral LTRs on transcription from internally placed
genes. Such effects include increased transcription (Jolly et at
(1983) Nucleic Acids Res. 11:1855-1872) or suppression of
transcription (Emerman and Temin (1984) Cell 39:449-467). This
strategy can also be used to eliminate downstream transcription
from the 3' LTR into genomic DNA (Herman and Coffin (1987) Science
236:845-848). This is of particular concern in human gene therapy
where it is of critical importance to prevent the adventitious
activation of an endogenous oncogene.
[0056] However, the plasmid vector used to produce the viral genome
within a host cell/packaging cell will also include transcriptional
regulatory control sequences operably linked to the lentiviral
genome to direct transcription of the genome in a host
cell/packaging cell. These regulatory sequences may be the natural
sequences associated with the transcribed lentiviral sequence, i.e.
the 5' U3 region, or they may be a heterologous promoter such as
another viral promoter, for example the CMV promoter. Some
lentiviral genomes require additional sequences for efficient virus
production. For example, in the case of HIV, rev and RRE sequence
are preferably included. However the requirement for rev and RRE
may be reduced or eliminated by codon optimisation. Further details
of this strategy can be found in WO 01/79518. Alternative sequences
which perform the same function as the rev/RRE system are also
known. For example, a functional analogue of the rev/RRE system is
found in the Mason Pfizer monkey virus. This is known as the
constitutive transport element (CTE) and comprises an RRE-type
sequence in the genome which is believed to interact with a factor
in the infected cell. The cellular factor can be thought of as a
rev analogue. Thus, CTE may be used as an alternative to the
rev/RRE system. Any other functional equivalents which are known or
become available may be relevant to the invention. For example, it
is also known that the Rex protein of HTLV-I can functionally
replace the Rev protein of HIV-1. It is also known that Rev and Rex
have similar effects to IRE-BP.
[0057] In a particularly preferred embodiment, the lentiviral
vector according to the present invention consists of a
self-inactivating minimal lentiviral vector, derived from Equine
Infectious Anaemia Virus (EIAV), preferably encoding three enzymes
that are involved in the dopamine synthetic pathway. The proteins
encoded by such a vector may comprise a truncated form of the human
tyrosine hydroxylase (TH*) gene (which lacks the N-terminal 160
amino acids involved in feedback regulation of TH), the human
aromatic L-amino-acid decarboxylase (AADC), and the human
GTP-cyclohydrolase 1 (GTP-CH1) gene. The vector may be produced by
the transient transfection of cells (e.g.HEK293T cells) with three
plasmids, encoding for: (1) the recombinant EIAV ProSavin.RTM.
(Oxford BioMedica plc, Oxford UK) vector genome (pONYK1-ORT, WO
02/29065 and Farley et at (2007) J. Gen. Med. 9:345-356); (2) the
synthetic EIAV gag/pol expression vector (pESGPK, WO 01/79518 and
WO 05/29065) and (3) the VSV-G envelope expression vector
(pHGK)
Packaging Sequence
[0058] As utilised within the context of the present invention the
term "packaging signal" which is referred to interchangeably as
"packaging sequence" or "psi" is used in reference to the
non-coding, cis-acting sequence required for encapsidation of
lentiviral RNA strands during viral particle formation. In HIV-1,
this sequence has been mapped to loci extending from upstream of
the major splice donor site (SD) to at least the gag start
codon.
[0059] As used herein, the term "extended packaging signal" or
"extended packaging sequence" refers to the use of sequences around
the psi sequence with further extension into the gag gene. The
inclusion of these additional packaging sequences may increase the
efficiency of insertion of vector RNA into viral particles.
Pseudotyping
[0060] Preferably, the lentiviral vector according to the present
invention has been pseudotyped. In this regard, pseudotyping can
confer one or more advantages. For example, with the lentiviral
vectors, the env gene product of the HIV based vectors would
restrict these vectors to infecting only cells that express a
protein called CD4. But if the env gene in these vectors has been
substituted with env sequences from other RNA viruses, then they
may have a broader infectious spectrum (Verma and Somia (1997)
Nature 389(6648):239-242). By way of examples, Miller et al.
pseudotyped an MoMLV vector with the envelope from the amphotropic
retrovirus 4070A (Mol. Cell. Biol. 5:431-437) other workers have
pseudotyped an HIV based lentiviral vector with the glycoprotein
from VSV (Verma and Somia (1997) Nature 389(6648):239-242).
[0061] In another alternative, the Env protein may be a modified
Env protein such as a mutant or engineered Env protein.
Modifications may be made or selected to introduce targeting
ability or to reduce toxicity or for another purpose (Marin et at
(1996) J Virol 70(5):2957-2962; Nilson et at (1996) Gene Ther
3(4):280-286; and Fielding et at (1998) Blood 91(5):1802-1809 and
references cited therein).
[0062] The vector may be pseudotyped, for example with a gene
encoding at least part of the rabies G protein or the VSV-G
protein.
[0063] VSV-G:
[0064] The envelope glycoprotein (G) of Vesicular stomatitis virus
(VSV), a rhabdovirus, is an envelope protein that has been shown to
be capable of pseudotyping certain retroviruses including
lentiviruses.
[0065] Its ability to pseudotype MoMLV-based retroviral vectors in
the absence of any retroviral envelope proteins was first shown by
Emi et al.(1991) J. Virol. 65:1202-1207). WO 94/294440 teaches that
retroviral vectors may be successfully pseudotyped with VSV-G.
These pseudotyped VSV-G vectors may be used to transduce a wide
range of mammalian cells. More recently, Abe et al. (1998) J. Virol
72(8): 6356-6361 teach that non-infectious retroviral particles can
be made infectious by the addition of VSV-G.
[0066] Burns et at (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037)
successfully pseudotyped the retrovirus MLV with VSV-G and this
resulted in a vector having an altered host range compared to MLV
in its native form. VSV-G pseudotyped vectors have been shown to
infect not only mammalian cells, but also cell lines derived from
fish, reptiles and insects (Burns et at (1993) Proc. Natl. Acad.
Sci. USA 90:8033-8037). They have also been shown to be more
efficient than traditional amphotropic envelopes for a variety of
cell lines (Yee et al.(1994) Proc. Natl. Acad. Sci. USA
91:9564-9568 and Emi et al. (1991) J. Virol. 65:1202-1207). VSV-G
protein can also be used to pseudotype certain lentiviruses and
retroviruses because its cytoplasmic tail is capable of interacting
with the retroviral cores.
[0067] The provision of a non-lentiviral pseudotyping envelope such
as VSV-G protein gives the advantage that vector particles can be
concentrated to a high titre without loss of infectivity (Akkina et
at (1996) J. Virol. 70:2581-2585). Lentivirus and retrovirus
envelope proteins are apparently unable to withstand the shearing
forces during ultracentrifugation, probably because they consist of
two non-covalently linked subunits. The interaction between the
subunits may be disrupted by the centrifugation. In comparison the
VSV glycoprotein is composed of a single unit. VSV-G protein
pseudotyping can therefore offer potential advantages.
[0068] WO 00/52188 describes the generation of pseudotyped
retroviral and lentiviral vectors, from stable producer cell lines,
having vesicular stomatitis virus-G protein (VSV-G) as the
membrane-associated viral envelope protein, and provides a gene
sequence for the VSV-G protein.
[0069] Ross River Virus
[0070] The Ross River viral envelope has been used to pseudotype a
nonprimate lentiviral vector (FIV) and following systemic
administration predominantly transduced the liver (Kang et at
(2002) J Virol 76(18):9378-9388.). Efficiency was reported to be
20-fold greater than obtained with VSV-G pseudotyped vector, and
caused less cytotoxicity as measured by serum levels of liver
enzymes suggestive of hepatotoxicity.
[0071] Ross River Virus (RRV) is an alphavirus spread by mosquitoes
which is endemic and epidemic in tropical and temperate regions of
Australia. Antibody rates in normal populations in the temperate
coastal zone tend to be low (6% to 15%) although sero-prevalence
reaches 27 to 37% in the plains of the Murray Valley River system.
In 1979 to 1980 Ross River Virus became epidemic in the Pacific
Islands. The disease is not contagious between humans and is never
fatal, the first symptom being joint pain with fatigue and lethargy
in about half of patients (Fields Virology Fifth Edition (2007)
Eds. Knipe and Howley. Lippincott Williams and Wilkins)
[0072] Baculovirus GP64
[0073] The baculovirus GP64 protein has been shown to be an
attractive alternative to VSV-G for viral vectors used in the
large-scale production of high-titre virus required for clinical
and commercial applications (Kumar M, Bradow B P, Zimmerberg J
(2003) Hum. Gene Ther. 14(1):67-77). Compared with
VSV-G-pseudotyped vectors, GP64-pseudotyped vectors have a similar
broad tropism and similar native titres. Because, GP64 expression
does not kill cells, 293T-based cell lines constitutively
expressing GP64 can be generated.
[0074] Rabies G
[0075] In the present invention the vector may be pseudotyped with
at least a part of a rabies G protein or a mutant, variant,
homologue or fragment thereof.
[0076] Teachings on the rabies G protein, as well as mutants
thereof, may be found in WO 99/61639 and well as Rose et al (1982)
J. Virol. 43:361-364, Hanham et al (1993) J. Virol. 67:530-542 ;
Tuffereau et al (1998) J. Virol. 72:1085-1091, Kucera et al (1985)
J. Virol. 55:158-162 ; Dietzschold et al (1983) PNAS 80:70-74; Seif
et al (1985) J. Virol. 53:926-934 ; Coulon et al (1998) J. Virol.
72:273-278 ; Tuffereau et al (1998) J. Virol. 72:1085-10910; Burger
et al (1991) J. Gen. Virol. 72:359-367 ; Gaudin et al (1995) J.
Virol. 69:5528-5534 ; Benmansour et al (1991) J. Virol.
65:4198-4203 ; Luo et al (1998) Microbiol. Immunol. 42:187-193,
Coll (1997) Arch. Virol. 142:2089-2097; Luo et al (1997) Virus Res.
51:35-41; Luo et al (1998) Microbiol. Immunol. 42:187-193; Coll
(1995) Arch. Virol. 140:827-851; Tuchiya et al (1992) Virus Res.
25:1-13; Morimoto et al (1992) Virology 189:203-216; Gaudin et al
(1992) Virology 187:627-632; Whitt et al (1991) Virology
185:681-688; Dietzschold et al (1978) J. Gen. Virol. 40:131-139;
Dietzschold et al (1978) Dev. Biol. Stand. 40:45-55; Dietzschold et
al (1977) J. Virol. 23:286-293 and Otvos et al (1994) Biochim.
Biophys. Acta 1224:68-76. A rabies G protein is also described in
EP 0445625.
[0077] Alternative Envelopes
[0078] Other envelopes which can be used to pseudotype lentiviral
vectors include Mokola, Ebola, 4070A and LCMV (lymphocytic
choriomeningitis virus).
[0079] Retroviral and lentiviral vectors have been proposed as a
delivery system for the transfer of a nucleotide of interest (NOI)
in vivo to one or more sites of interest.
[0080] The expression products encoded by the NOIs may be proteins
which are secreted from the cell. Alternatively the NOI expression
products are not secreted and are active within the cell.
[0081] The or each NOI may be prophylactically, therapeutically
and/or diagnostically relevant to a neurological disorder. Suitable
NOIs include, but are not limited to: sequences encoding enzymes,
cytokines, chemokines, hormones, antibodies, anti-oxidant
molecules, engineered immunoglobulin-like molecules, a single chain
antibody, fusion proteins, immune co-stimulatory molecules,
immunomodulatory molecules, anti-sense RNA, microRNA, shRNA, siRNA,
ribozymes, a transdomain negative mutant of a target protein, a
toxin, a conditional toxin, an antigen, a tumour suppresser protein
and growth factors, membrane proteins , vasoactive proteins and
peptides, anti-viral proteins and ribozymes, and derivatives
thereof (such as with an associated reporter group). The NOIs may
also encode pro-drug activating enzymes.
[0082] In the present invention, the NOI can be, for example, a
synthetic RNA/DNA sequence, a recombinant RNA/DNA sequence (i.e.
prepared by use of recombinant DNA techniques), a cDNA sequence or
a partial genomic DNA sequence.
[0083] The NOI may be useful in the treatment of a
neurodegenerative disorder, for example Parkinson's disease.
[0084] The NOI may encode an enzyme or enzymes involved in dopamine
synthesis or storage. For example, the enzyme may be one or more of
the following: Tyrosine Hydroxylase (TH), GTP-cyclohydrolase I
(GTP-CH1) and/or Aromatic Amino Acid Dopa Decarboxylase (AADC). The
sequences of all three genes are available: Accession Nos. X05290,
U19523 and M76180 respectively.
[0085] Alternatively the NOI may encode the vesicular monoamine
transporter 2 (VMAT2, Accession number L23205.1). The viral genome
may comprise an NOI encoding AADC and an NOI encoding VMAT 2. Such
a genome may be used in the treatment of Parkinson's disease, in
particular in conjunction with peripheral administration of
L-DOPA.
[0086] Alternatively the NOI may encode a growth factor capable of
blocking or inhibiting degeneration in the nigrostriatal system or
which prevents TH-positive neurones from dying, or which stimulates
regeneration and functional recovery. For example the NOI may
encode glial cell-line derived neurotrophic factor (GDNF),
brain-derived neurotrophic factor (BDNF), nerve growth factor
(NGF), persephin growth factor, artemin growth factor, or neurturin
growth factor, cilliary neurotrophic factor (CNTF), neurotrophin-3
(NT-3), neurotrophin-4 (NT-4), pantropic neurotrophin, acidic
fibroblast growth factor (aFGF), basic fibroblast growth factor
(bFGF), interleukin-1 beta (IL-1.beta.), tumor necrosis factor
alpha (TNF-.alpha.), insulin growth factor-2, VEGF-A, VEGF-B,
VEGF-C/VEGF-2, VEGF-D, VEGF-E, PDGF-A, PDGF-B, hetero- and
homo-dimers of PDFG-A and PDFG-B and other related or unrelated
neurotrophic factors. The lentiviral vector may comprise one or
more of these NOIs encoding neurotrophic factors.
[0087] The NOI may also encode an anti-angiogenic protein or
anti-angiogenic proteins selected from the group consisting of
angiostatin, endostatin; platelet factor 4, pigment epithelium
derived factor (PEDF), restin, interferon--alpha,
interferon-inducible protein, gro-beta and tubedown-1,
Interleukin(IL)-1, IL-12, retinoic acid, anti-VEGF antibodies,
aptamers, antisense oligos, siRNA, thrombospondin, VEGF receptor
proteins such as those described in U.S. Pat. No. 5,952,199 and
U.S. Pat. No. 6,100,071, and anti-VEGF receptor antibodies.
[0088] The NOI may encode all or part of the protein of interest
("POI"), or a mutant, homologue or variant thereof. For example,
the NOI may encode a fragment of the POI which is capable of
functioning in vivo in an analogous manner to the wild-type
protein.
[0089] One of the NOIs may comprise a truncated form of the TH
gene, lacking the regulatory domain. Such an NOI avoids feed-back
inhibition by dopamine which may limit expression of the
full-length enzyme.
[0090] The term "mutant" includes POIs which include one or more
amino acid variations from the wild-type sequence. For example, a
mutant may comprise one or more amino acid additions, deletions or
substitutions. A mutant may arise naturally, or may be created
artificially (for example by site-directed mutagenesis).
[0091] Here, the term "homologue" means an entity having a certain
homology with the NOI, or which encodes a protein having a degree
of homology with the POI. Here, the term "homology" can be equated
with "identity".
[0092] In the present context, a homologous sequence may be at
least 75, 85 or 90% identical, or at least 95 or 98% identical to
the subject sequence at the amino acid or nucleotide level.
Typically, the homologues will comprise or encode the same active
sites etc. as the subject sequence.
[0093] A number of NOIs may be used in combination. If the
lentiviral vector comprises two or more NOIs, in order for both of
the NOIs to be expressed, there may be two or more transcription
units within the vector genome, one for each NOI. 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, so it is preferable to use one or more
internal ribosome entry site(s) (IRES) to initiate translation of
the second (and subsequent) coding sequence(s) in a poly-cistronic
message (Adam et al 1991 J. Virol. 65:4985). An example of such
vectors is described in WO 02/29605.
Pharmaceutical Composition
[0094] The lentiviral vector of the present invention may be
provided in the form of a pharmaceutical composition. The
pharmaceutical composition may be used for treating an individual
by gene therapy, wherein the composition comprises a
therapeutically effective amount of the lentiviral vector.
[0095] The viral preparation may concentrated by
ultracentrifugation. WO 2009/153563 describes methods for the
downstream processing of lentiviral vectors. The resulting
pharmaceutical composition may have at least 10.sup.7 T.U./mL, for
example from 10.sup.7 to 10.sup.9 T.U./mL, or at least 10.sup.9
T.U./mL. (The titer is expressed in transducing units per mL
(T.U./mL) as titred on a standard D17 of HEK293T cell lines).
[0096] The pharmaceutical composition may be used to treat a human
or animal, for example a primate animal subject or a companion
animal subject.
[0097] The composition may optionally comprise a pharmaceutically
acceptable carrier, diluent, excipient or adjuvant. The choice of
pharmaceutical carrier, excipient or diluent can be selected with
regard to the intended route of administration and standard
pharmaceutical practice. The pharmaceutical compositions may
comprise as (or in addition to) the carrier, excipient or diluent,
any suitable binder(s), lubricant(s), suspending agent(s), coating
agent(s), solubilising agent(s), and other carrier agents that may
aid or increase the viral entry into the target site (such as for
example a lipid delivery system).
Diseases
[0098] The lentiviral vector used in the present invention is for
use in treating a neurological condition. For example, the vector
may be useful for the treatment and/or prevention of
neurodegenerative diseases.
[0099] Diseases which may be treated include, but are not limited
to: Parkinson's disease; amyotrophic lateral sclerosis (motor
neurone disease); Huntington's disease, and disorders of movement,
such as Friedreich's ataxia, cerebellar ataxia, distonias,
repetitive motion disorders, restless leg syndrome, tremor and
myoclonus; Alzheimer's disease and Pick's disease; stroke; focal
and generalised or idiopathic epilepsy; chronic pain, including
paresthesias, back pain, and diabetic neuropathy; brain tumours;
chronic fatigue syndrome; Creutzfeldt-Jakob disease (CJD) and
variant CJD; leukodystrophies, including Tay-Sachs disease, and
Wilson's disease; changes to intracranial pressure; cluster
headaches and migraine; multiple sclerosis; chronic eating
disorders including Prader-Willi disorder; schizophrenia; affective
disorders; mania and sleeping disorders including sleep apnea.
[0100] In particular, the present invention is useful in treating
and/or preventing Parkinson's disease.
[0101] Treatment by gene therapy with vectors capable of
delivering, for example, TH, GTP-CH1 and optionally AADC or AADC
and VMAT2, is likely to be particularly useful for the late stages
of PD patients which do not respond significantly to L-dopa
treatment. Treatment using AADC or AADC and VMAT2, in combination
with L-dopa administered peripherally may also be useful for late
stage PD patients.
Administration
[0102] The lentiviral vector used in the present invention is
administered to the brain, for example by injection into the
caudate putamen.
[0103] The vector may be administered via one, two, three, four,
five, six or more tracts per hemisphere.
[0104] The term cannula as used herein shall include cannulas,
catheters, needle or any other suitable device for the delivery of
therapeutics directly to the brain. WO 2008/100930, WO 2008/144585
and WO 2009/101397 describe such cannulas which could be used.
[0105] In a previously described administration system for a
lentiviral vector (Jarraya et at (2009) Sci Transl Med 14: 1(2)
2-4), the vector composition was administered in a discontinuous or
"punctate" fashion, by administering an aliquot (4 .mu.L) at the
bottom of the tract, withdrawing the needle a little way, then
administering a second aliquot (3 .mu.L) and withdrawing the needle
a little further, (second time); then administering a third aliquot
(3 .mu.L); thus aliquots had been deposited at 3 points along each
needle tract delivering a total of 10 .mu.L.
[0106] Disadvantages associated with this system include the fact
that it is very slow and labour-intensive, and that, as there are 5
needle tracts per hemisphere with 3 administration sites per tract,
there are fifteen potential sites for tissue damage and
inflammation in each hemisphere. Because of this, it is not
possible to increase the dose of vector administered to each
hemisphere using this method of vector delivery as it would
increase the surgery time significantly.
[0107] In the method of the present invention, the vector may be
delivered at one deposit point for each tract and larger volumes
can be delivered via each tract. In the method of the invention,
the vector composition is continuously infused. Continuous
administration at a single point overcomes the disadvantages
mentioned above in connection with the previous system. Using this
method, higher doses of vector can be administered to each
hemisphere within a practical surgery time.
[0108] The term "continuous infusion" means that infusion of the
vector composition does not stop and the needle is not moved during
delivery. For a given cannula, the entire volume of vector
composition to be delivered is administered at a single deposit
point and in one "push".
[0109] During continuous infusion, the flow rate of the vector
composition may be substantially constant, gradually increased or
increased in a stepwise manner.
[0110] Delivery "directly to the brain" means that the lentiviral
vector is administered directly to brain tissue using an invasive
procedure such as injection. The lentiviral vector may be delivered
to the putamen, for example the motor putamen.
[0111] During delivery the volume of lentiviral vector delivered
via each cannula tract may be 10-600 .mu.L, or about 40-200 .mu.L
may be delivered per tract. One to six tracts may be used for each
hemisphere.
[0112] The vector may be delivered using a cannula of sufficiently
narrow bore to prevent substantial backflow of the vector
composition. For example, the cannula may be of a bore such that
less than 20%, 15%, 10% or 5% of the vector composition flows back
up the needle during or after delivery.
[0113] The vector may be delivered using a cannula having an outlet
equal to or narrower in diameter than a 23-gauge needle. The outlet
may be about 28-gauge. The device may have an outlet of less than
23-gauge and more than 33-gauge.
[0114] The internal diameter of the cannula may be may be less that
0.35, 0.3, 0.35, 0.2 or 0.15 mm.
[0115] The present invention also provides a method for improving
distribution of a lentiviral vector in the medio-lateral and
dorso-ventral axes of the putamen when administered directly to the
brain of a subject, by delivering the lentiviral vector using a
continuous infusion for each cannula tract.
[0116] Adaptation of an existing method in order to improve
distribution of the lentiviral vector in the medio-lateral and
dorso-ventral axes of the putamen and to allow dose escalation may
involve any or all of the following: reduction in the number of
deposit point per tract; using continuous infusion of the
lentiviral vector composition; creating and/or maintaining a
pressure gradient during interstitial infusion; using a higher flow
rate; and/or delivering a larger volume.
[0117] Typically a lentiviral vector is delivered using a cannula
or other injection device which is inserted into the brain tissue
in the chosen subject. When delivering ProSavin.RTM. for the
treatment of Parkinson's disease, the striatum is a suitable area
of the brain to target. Other areas would be suitable for the
treatment of other neurological disorders. One skilled in the art
could readily determine which general area of the CNS would be an
appropriate target. Stereotactic maps and positioning devices are
available. Positioning may also be conducted by using anatomical
maps obtained by CT and/or MR imaging of the subject's brain to
help guide the injection device to the chosen target area.
Fluid Convection
[0118] In a previously described administration system for a
lentiviral vector (Jarraya et at (2009) as above), the vector
composition penetrated the brain parenchyma by diffusion. Diffusion
depends on the free concentration gradient along with the
diffusivity of the agent. Dispersal by diffusion may result in poor
penetration, particularly for high molecular weight agents such as
lentiviral vectors.
[0119] The diffusion of therapeutics within the extracellular space
is necessary to enable such therapeutics to access the target
tissues such as neuronal and glial cells. Thorne and Nicholson
(Thorne and Nicholson (2006) PNAS 104; 5567-5572) have determined
that the normal extracellular space is between 38 and 64 nm. They
suggest that nanoparticle delivery systems of greater than 100 nm
will be too large to transit the normal extracellular space. As
stated above lentiviral vectors are viral-based nanoparticle
delivery systems with diameters of around 100 nm.
[0120] In the method of the invention, the lentiviral vector
solution is dispersed by fluid convection. Interstitial infusion is
performed using a high flow rate which the inventors have shown to
greatly enhance the distribution of the lentiviral vector.
[0121] The flow rate may be maintained at a steady state during
continuous infusion of the vector, or it may be increased in order
to maintain the convection pressure (see below). An increasing
pressure gradient may be, for example, steadily increasing or it
may follow a ramped procedure characterised by several small
step-wise increases of flow rate during the continuous
infusion.
[0122] Fluid pressure may be maintained, gradually increased or
ramped during delivery using systems known in the art, such as a
programmable osmotic, infusion or other pump known to one skilled
in the art. The high flow rate is maintained for the entire
infusion process, delivering the complete volume of vector (e.g. 10
to 600 .mu.L).
[0123] Infusion pressure is a function of flow rate, viscosity of
lentiviral vector preparation, and cannula size. The infusion
pressure should be selected such that it provides a sufficient
pressure gradient to enhance distribution of the lentiviral vector,
but insufficient to a) cause significant damage to the tissue
surrounding the administration site; and/or b) cause "back-flow"
and leakage of the solution out of the cannula tract.
[0124] In the method of the present invention, the lentiviral
vector may be infused into the brain at a flow rate of at least 1.0
.mu.L/min, 1.5 .mu.L/min, or 2 .mu.L/min, or between 1-2 .mu.L/min,
between 1-3 .mu.L/min, between 2-6 .mu.L/min, between 2-4 .mu.L/min
or between 2.5-3.5 .mu.L/min. The lentiviral vector may be infused
into the brain at a flow rate of about 3 .mu.L/min.
[0125] During infusion, the convection pressure (which is the
difference between infusion pressure and intracranial pressure) may
decrease as the total volume delivered increases. In order to
maintain convention pressure, the flow rate may be steadily
increased during administration. If such a system is employed, the
flow rates mentioned in the preceding paragraph may represent the
starting flow rates.
Kit
[0126] The present invention also provides a kit for delivering a
lentiviral vector directly to the brain of the subject, which
comprises one to twelve cannulas. Cannulas may be re-used for
multiple tracts for each patient or a new cannula may be used for
each tract.
[0127] The vector may be delivered for example, using a cannula or
catheter or a needle (such as a Hamilton needle). The term cannula
as used herein shall include cannulas, catheters, needle or any
other suitable device for the delivery of therapeutics directly to
the brain. WO 2008/100930, WO 2008/144585 and WO 2009/101397
describe such cannulas which could be used.
[0128] The cannula may be pre-filled with the lentiviral vector
composition. The cannula may be primed prior to positioning within
the brain.
[0129] The delivery device may, for example, be a stainless steel
28-gauge Hamilton syringe or a fused silica 28-gauge infusion
cannula.
[0130] If the kit is for use in a method employing one delivery
tract per hemisphere, the cannula may, for example, be capable of
delivering between about 40 .mu.L, 100 .mu.L, 150 .mu.L, 200 .mu.L,
300 .mu.L, 400 .mu.L, 500 .mu.L or 600 .mu.L of the lentiviral
vector. If the kit is for use in a method employing two delivery
tracts per hemisphere, the kit may comprise four cannulas.
Alternatively a single cannula may be re-used for each tract, or
one cannula may be used to deliver vector to each hemisphere, in
which case it would be filled with the lentiviral vector
composition and primed before each insertion into the target
area.
[0131] The cannula may also be pre-connected or adapted for
connection to an infusion device such as a pump described above.
Alternatively the cannula may be attached to known systems for
vector administration, such as a syringe which is controlled by a
micropump such as those distributed by World Precision Instruments,
or the microinjector system (Kopf, USA). Such a delivery system may
be suitable for use with a stereotactic frame.
[0132] In a further aspect of the invention the narrow bore
delivery devices used in connection with the method the invention
may be implanted in a subject, to remain over a substantial period
of time extending at least several hours to days or more, thus
allowing infusion of vector to take place outside of an operating
room. It may be desirable that such devices are sufficiently
resilient that they can be secured with a fixation device to
prevent movement away from the target without damaging the cannula
infusion system. It is also desirable that such devices can be
imaged to confirm the location in the brain and to ensure that the
catheter has not moved from the target at any point.
[0133] Narrow bore delivery devices suitable for use in the present
invention may have a sufficiently small diameter at the distal,
infusion end, to minimize local tissue trauma, with a very small
dead space yet with the strength of a larger catheter to prevent
breakage and permit fixation, and with the ability to visualize the
catheter by imaging.
[0134] As will be known to those skilled in the art, there are a
number of narrow bore delivery devices that may be suitable for use
in the present invention (for example, see the delivery device
described in WO 2007/044023).
[0135] The narrow bore infusion device of the invention is
optionally attached to the head of the subject. Potential fixation
methods include, but are not limited to, fixation to the skull near
the burr hole with a metal plate, such as a titanium plate,
fixation with a plastic capping system placed within the burr hole,
or externalization through the scalp and suturing of the guide
catheter to the scalp.
[0136] The kit may also comprise instructions for storage and/or
use.
[0137] The invention will now be further described by way of
Examples, which are meant to serve to assist one of ordinary skill
in the art in carrying out the invention and are not intended in
any way to limit the scope of the invention.
EXAMPLES
Example 1
Comparing Distribution of a Lentiviral Vector in the Putamen
Following a) Conventional Delivery and b) Delivery Using a Single
Site and a High Flow-Rate
[0138] Administration of EIAV-LacZ vector suspension using the
previously described technique (50 .infin.L total volume vector
administered to the putamen through 5 needle tracts using a
Hamilton syringe and 23-gauge needle, each delivering 10 .mu.L per
tract (via three deposits) provided a moderate spread of vector in
the dorso-ventral axis of the putamen that reached between 40 and
50% the depth of this brain structure (FIG. 1).
[0139] Spread of vector in the medio-lateral axis was very much
less than with the dorso-ventral axis, with the vector only
spreading around 1-2 mm at the greatest point. However, due to the
positioning of the five needle tracts along the axis of the
putamen, spread of vector along the rostro-caudal axis was around
7.8 mm in both of the two hemispheres that were administered vector
by this paradigm.
[0140] In contrast, using the method of the present invention
(infusing the entire 50 .mu.L of vector suspension at one site in
the putamen through the 23-gauge needle with a Hamilton syringe or
using a 28-gauge fused silica cannula at either 1 or 3 .mu.L/min)
produced improved vector spread in the medio-lateral and
dorso-ventral axis compared with the five tract regimen with the
greatest degree of spread observed with the 28-gauge cannula at
either 1 or 3 .mu.L/min flow rates.
[0141] Vector delivery of 50 .mu.L vector using a single tract of
delivery with the 28-gauge cannula device resulted in a similar
rostro-caudal spread than with the five tract paradigm at both flow
rates (6.2-8.6 mm spread for the 28-gauge cannula and 7.8 mm spread
with the five tracts and discontinuous flow-rate). A lower
rostro-caudal spread was observed with a single infusion using the
23-gauge needle and Hamilton syringe (5.0-6.2 mm spread).
[0142] When the total volume of vector distribution was compared it
was surprisingly found that the single administration of 50 .mu.L
of vector using a 23-gauge delivery device was less than the
five-tract delivery method. Even more surprising, the 28-gauge
delivery device gave a greater volume of vector distribution than
the five-tract method--more than twice the volume with the higher
flow rate. This increase in volume was at least partially thought
to result from reduced backflow of vector along the needle tract
using this narrow bore delivery device.
[0143] Overall these observations were consistent with the vector
spreading evenly from the tip of the device when a single infusion
was performed with the 28-gauge cannula. Conversely, with the 5
tract delivery approach and to a less extent with the single
infusion using the 23-gauge needle and syringe, the distribution of
vector was more confined to the area directly around the needle
tract. There was no evidence for damage to surrounding neuronal
tissue or rupture of the blood-brain barrier as a result of the
infusions in all groups. A 33-gauge plastic cannula was also
evaluated but was considered too flexible to be used for routine
surgery potentially contributing to the failure of the surgeon to
place the cannula at the correct site within the putamen. The
28-gauge fused silica cannula resulted in a similar spread of
vector using both flow rates (1 or 3 .mu.L/min). The finer gauge
28-gauge cannula resulted in less scarring than the 23-gauge
Hamilton needle. A similar number of cells (>90%) were doubly
stained for .beta.-galactosidase and for NeuN irrespective of the
delivery paradigm, confirming neuronal targeting of the vector in
all cases.
[0144] The local inflammatory response to infusion of EIAV-LacZ
vector was assessed in this study by histological evaluation of
inflammatory markers CD4 and CD8 (T-cells) and CD68 (activated
microglia). Positive staining for each of the markers was observed
in the vicinity of the needle tract for all of the methods of
delivery and injection devices, however, there was very little
vector-associated inflammatory response and no differences between
the administration methods were observed. This result indicates
that the there is no increased inflammatory response associated
with vector delivery using a single infusion at a higher flow.
[0145] No overt toxicity or abnormal clinical signs to vector
administration was observed in any of the study animals. Animals
did not display any abnormal locomotor activity or behaviour and
were observed to feed normally.
TABLE-US-00001 TABLE 1 Summary of vector distribution using
different vector delivery parameters Rostro-caudal spread Delivery
Parameters of vector (mm) 50 .mu.L delivered through 5 tracts using
a 23-gauge 7.8 and 7.8 Hamilton syringe 1 .mu.L/min Single 50 .mu.L
infusion using a 23-gauge Hamilton 5.8 and 6.2 syringe, 1 .mu.L/min
Single 50 .mu.L infusion using a 23-gauge Hamilton 5.0 syringe, 3
.mu.L/min Single 50 .mu.L infusion using a 28-gauge fused silica
8.6 and 6.2 cannula, 1 .mu.L/min Single 50 .mu.L infusion using a
28-gauge fused silica 7.0 and 6.2 cannula, 3 .mu.L/min Single 100
.mu.L infusion using a 28-gauge Hamilton syringe, 3 .mu.L/min
Single 100 .mu.L infusion using a 28-gauge fused silica cannula, 3
.mu.L/min
TABLE-US-00002 TABLE 2 Data from a single experiment showing the
distribution volume. Volume Injection A-P of distri- Injection
volume/ Flow distribution bution Injection Device method Rate (mm)
(mm.sup.3) Stainless steel 50 .mu.L vector delivered 1 .mu.L/min
7.8 35.1 needle and glass through 5 injection syringe (23- tracts
with three gauge) deposits per tract Stainless steel 50 .mu.L,
single infusion 1 .mu.L/min 5.8 10.0 needle and glass 3 .mu.L/min
5.0 25.7 syringe (23- gauge) Fused silica 50 .mu.L, single infusion
1 .mu.L/min 8.6 60.2 cannula (28- 3 .mu.L/min 7.0 91.7 gauge)
CONCLUSION
[0146] Previously, small volumes of lentiviral vectors have been
delivered to the brain via multiple deposits within each cannula
tract using slow flow rates. Such a method is cumbersome and has
not enabled dose escalation studies. A simplified continuous
infusion paradigm that allows the administration of greater volumes
of vector at a faster flow rate was considered to be more
applicable to surgical employment in the treatment of various
neurological conditions.
[0147] It was not known whether vectors as large as lentiviruses
could be effectively distributed into the brain through a single
infusion paradigm as these vectors are considerably larger that the
AAV particles which have been examined previously in primate brain
(Bankiewicz et al (2000) Exp. Neurol. 164:2-14; Hadaczek et al
(2006) Hum. Gene Ther 17:291-302 and Hadaczek et at (2009) Hum.
Gene Ther 20:229-237). As the diameter of lentiviral vectors
(around 100 nm) is larger than the extracellular space (38-64 nm),
it is possible that lentiviral vector particles would not have been
effectively driven through the extracellular space, which may be
prohibitive for particles as large as the lentiviral vectors. It
is, however, clear that lentiviral vectors such as EIAV vectors can
be effectively driven through the putamen region using a continuous
infusion method that is easily controlled and which provides a
volume of transduction that is better than spotting the vector
around the target region using multiple cannula placements.
[0148] Previous studies in rodents have reported that at low flow
rates and during the CED process, vector can spread through the
perivascular space, possibly driven by the pulsatile action of
blood flow through the brain capillary network (Hadaczek et al
(2004) Hum Gene Ther. 15:469-79). However, we have found little
evidence of this phenomenon when the flow rate was maintained at a
high level. Restricting this perivascular transport may reduce
vector distribution to non-target areas.
[0149] There has been concern that some viral vectors may pose a
risk of central inflammation and that infusing vectors, at a single
flow rate, or through the process of CED may exacerbate an
inflammatory response. It has been recently demonstrated that the
AAV serotype-1 vectors, which have been examined as carriers of
therapeutic genes, produced a robust humoral and cellular response
when administered to the striatum and cortex of primates (Hadaczek
et at (2009) Hum. Gene Ther 20:229-237). No evidence for an
increased local imflammatory response has been observed with a
single infusion of an EIAV-based vector using a 28-gauge cannula
with administration flow rates of between 1 and 3 .mu.L/min.
[0150] In conclusion, these studies indicate that therapeutic genes
aimed at treating various neurological conditions may be safely
administered by the EIAV vector to target brain regions using the
simplified infusion paradigm and that a wide volume of target cells
can be effectively transduced.
Methods and Materials
Animals
[0151] Six male and six female cynomolgus macaques were housed in
single sex compatible groups during their acclimatisation period at
constant temperature (22 .+-.2.degree. C.) and humidity (45%-65%)
under a 12 hour light/dark cycle (lights on 07:30) prior to vector
administration. The animals were then singly housed following
surgical administration of vector in individual stainless steel
boxes of standard dimensions of 1.10 m.sup.2 floor surface.times.1
m high. Air in the animal room was changed approximately 10 times
per hour. Animals were fed Old World Monkey pellets (SDS DIETEX
#808004) and had access to tap water ad libitum. Animals use and
care was administered to the Directive 86/609/EEC European
Convention for the Protection of Vertebrate Animals used for
Experimental and Other Scientific Purposes and the Animals
(Scientific Procedure) Act 86 for the United Kingdom.
Vector Administration
[0152] Animals were fasted prior to surgery and water prevented
access to water for approximately 6 hours. The animals were
administered atropine sulphate (0.04 mg/kg, i.m.), ketamine HCl (10
mg/kg, i.m.) and buprenorphine (0.01 mg/kg, i.m.) and then
anaesthetised with propofol (3 mg/kg i.v.) 10 min later. Animals
were also injected with amoxicillin (30 mg/kg, s.c.) 24 hours prior
to surgery and then every 48 hours up to one week after the
surgery. The animals were inturbated and maintained in anaesthesia
with isoflurane inhalant anaesthetic delivered through a
volume-regulated respirator. The ECG, O.sub.2 saturation and heart
rate was monitored and recorded. Body temperature was monitored
throughout the surgery using a rectal thermometer. The animals were
placed in a stereotaxic frame (Unim'ecanique, France) and vector
administered into the putamen using the microinjector system (Kopf,
USA) for administration of vector as single small deposits in the
putamen using a 6.5 mm long 23-gauge stainless steel Hamilton
needle attached to a 710 Hamilton syringe (Hamilton Bonaduz AG,
Bonaduz, Switzerland). For the single putamen infusions, vector
suspensions were infused using either 6.5 mm long 23-gauge
stainless steel Hamilton needle and syringe, 6.5 mm long 28-gauge
stainless steel Hamilton needle and syringe or 6.5 mm long fused
silica injection cannula attached to a Hamilton syringe by a short
piece of plastic tubing to minimise the dead volume (Plastics-1,
Roanoke, Va., USA). After drilling a hole in the skull without
damaging the dura mater, a ventriculographic cannula mounted on a
glass syringe was introduced into the anterior horn of the lateral
ventricle and a contrast medium (Omnipaque, Nycomed, Norway)
injected. A stereotaxic atlas was used for precise adjustment
before insertion into the skull (Martin & Bowden (1996)
Neuroimage 4(2):119-150). Accurate position of the anterior
commissura (AC) was be deduced from ventriculography which was then
used to position both left and right putamen. The coordinates for
placement of the infusion cannulas was AC-1 mm (directly centre of
the motor area of the putamen). For multiple deposits within each
putamen, vector was injected at 3 depths delivering 4 mL at the
deepest deposit and then a further two deposits of 3 mL each
delivered 1 mm above one another to deliver 10 mL per needle tract.
The first of five Hamilton needle tracts was at the level of the
anterior commissure (AC), i.e. AC 0 mm, the second injection: 2 mm
caudal to the AC (AC -2 mm), third injection: 3 mm caudal to the AC
(AC -3 mm), forth injection: 5 mm caudal to the AC (AC -5 mm), and
fifth injection: 6 mm caudal to the AC (AC -6 mm) to distribute
vector along the rostro-caudal length of the putamen. The Hamilton
syringe and infusion catheter will be left in situ for an
additional 2 minutes after each injection/infusion before being
removed.
[0153] Vector was infused at a single flow rate of 0.5, 1 or 3
.mu.L/min using an a programmable small infusion pump
(UltraMicroPump III; World Precision Instruments, Sarasota, Fla.,
USA). Vector was administered bilaterally into the putamen in a
total volume of 50 .mu.L per hemisphere with each hemisphere
receiving vector delivered by a different flow rate or cannula
device. In one animal, TSSM formulation buffer was infused into the
putamen, in one hemisphere using the 23-gauge Hamilton needle
delivering buffer at 1 mL/min and in the other hemisphere the
vector was delivered by a 28-gauge Hamilton needle at 1 mL/min. The
TSSM-infused animal was used to assess the relative extend on
neuronal damage and inflammation response to sterile formulation
buffer alone, in the absence of viral vector. surgical records.
Following surgery, the animals were closely observed and kept warm
until they had regained the righting and swallowing reflexes before
being returned to their cages. Buprenorphine analgesia was
administered twice daily, for 3 days (0.02 mg/kg, i.m.) and body
weights were recorded weekly after surgery.
Histology
[0154] Animals were necropsied 4 weeks after dosing and following
an overnight fast through injection of ketamine (10 mg/kg) and then
euthanasia using sodium pentobarbital anaesthesia (between 10-30
mg/kg, i.v.). Following collection of a blood sample for assessment
of the immune response to the virus, the animals were exsanginated,
perfused with heparinized phosphate buffered saline (0.9% w/v
sodium chloride, USP containing approximately 25 U/mL heparin)
followed by a solution of 4% buffered paraformaldehyde in phosphate
buffered saline (PBS). The brains (with the dura removed) were
carefully dissected and placed in fresh 4% paraformaldehyde
overnight at 5.+-.3.degree. C. and then transferred to cold
filtered 30% sucrose solution in PBS for between 2 and 4 days. The
brains were then bisected down the midline into two hemispheres and
each hemisphere snap frozen in cold isopentane (-40 to -50.degree.
C.) and stored at -80.degree. C. before sectioning at forty micron
(40 .mu.m) thickness using a cryostat. Brain sections containing
the putamen region were collected in pots containing 5 sections
equating a distance of two hundred microns (200 .mu.m) travel in
the rostro-caudal axis of brain hemisphere. Free-floating brain
sections were stained with anti-.beta.-galactosidase monocloncal
antibodies. Secondary and tertiary antibodies were obtained from
the Vectastain anti mouse ABC kit (#PK-6102) and the methodology
was as per kit instructions. DAB visualisation was achieved using a
DAB peroxidise substrate kit (catalogue number SK-4100). Sections
were then mounted on glass microscope slides. Alternative brain
hemisphere sections were stained with anti-GFAP antibody (Chemicon
#MAB3402) at a concentration of 1:200. Selected sections were also
stained using H&E and the sections analysed microscopically to
assess levels of cell infiltrates and changes in tissue morphology.
Selected sections in the region of vector infusion were also
stained for the immunological markers CD8, CD4 and CD68 after
removing non-specific staining with 10% hydrogen peroxide followed
by an overnight incubation in 10% serum with 0.5% triton X-100.
Primary antibodies (IgG negative control, no primary negative
control, mouse anti human CD4, CD8 and CD68) were applied for two
hours only. After three 5 minute washes in PBS+0.02% Tween 20 the
anti-mouse vector elite ABC kit was used with three 5 minute washes
in between each step. A DAB peroxidase kit was then used for
visualisation.
[0155] The volume of vector distribution was calculated by
measuring the area of positive .beta.-galactosidase staining in
serial brain sections throughout the injected putamen and applying
standard stereology methods (using Cavalieri's principle) to
estimate total volume coverage.
Example 2
Comparing Efficacy of a Lentiviral Vector for Parkinson's Disease
Following Administration to the Motor Putamen of Parkinson's
Disease Patients Using a) Conventional Delivery and b) Delivery
Using a Reduced Number of Injection Sites with a Narrower Gauge
Device and a High Flow-Rate
[0156] A phase I/II clinical trial is ongoing to evaluate the
safety and efficacy of a lentiviral vector based treatment for
Parkinson's disease, called ProSavin.RTM.. ProSavin.RTM. is an EIAV
lentiviral vector that contains three genes which encode for
enzymes in the dopamine biosynthesis pathway. As part of the trial
the therapeutic potential of ProSavin.RTM. to correct symptoms of
Parkinson's disease was evaluated using 1) the conventional method
of administration and 2) a continuous method of infusion. The
conventional method involved injection of a total volume of 125
.mu.L of vector administered to each putamen through 5 needle
tracts using a Hamilton syringe and 23-gauge needle and an
administration rate of 1 .mu.L/min. A total of 25 .mu.L of vector
was administered along each injection tract with 5 deposits of 5
.mu.L of vector distributed along the tract. In the continuous
method of delivery the same total volume of vector was administered
(12 .mu.L) using a Hamilton syringe and a narrower 28-gauge
needle.
[0157] Three injections were performed into each putamen and a
continuous infusion of vector was made at each injection site using
an increased administration rate of 3 .mu.L/min. The volumes of
vector delivered were 42 .mu.L, 42 .mu.L and 43 .mu.L at the three
injection sites. The surgical time for vector administration using
the infusion method was almost half of that compared to the
conventional method.
[0158] The surgical procedures were safe and well tolerated in all
patients with both administration methods. There were no serious
adverse events reported in any patients relating to either
ProSavin.RTM. or the two administration methods.
[0159] The primary efficacy endpoint of the study was improvement
in the motor part (part III) of the Unified Parkinson's Disease
Rating Scale (UPDRS) at 6 months post treatment, compared with
baseline scores. A summary of improvements in motor function to
date, is shown in Table 3 (motor function is assessed according to
the Unified Parkinson's Disease Rating Scale [UPDRS] in patients'
"OFF" state, i.e. after withdrawal of Parkinson's disease
medication). In the group of patients receiving ProSavin.RTM. using
the conventional, discontinuous, 5-deposit/tract method an
improvement of 34% in UPDRS part III scores was observed at 6
months. Interestingly, patients that received the same total volume
of vector using the continuous infusion method showed a greater
improvement in UPDRS part III scores, reaching a 43% improvement at
6 months post treatment.
TABLE-US-00003 TABLE 3 Adminis- 3 6 1 2 tration months months. year
years Cohort.sup.2 Dose method (UPDRS) (UPDRS) (UPDRS) (UPDRS) 1,
1.times. Conven- Mean Mean Mean Mean n = 3 tional 27% 30% 29% 20%
Max. up Max. up Max. up Max. up to 30% to 50% to 44% to 30% 2,
2.times. Conven- Mean Mean Mean -- n = 3 tional 28% 34% 29% Max. up
Max. up Max. up to 53% to 53% to 56% 2b, 2.times. Contin- Mean Mean
-- -- n = 3 uous 26% 43% Infusion Max. up Max. up to 52% to 61%
[0160] In addition patients showed an average improvement of 26% in
UPDRS part III "ON" score at 6 months. Patient diary data showed an
increase in functional improvement in the time oral L dopa was
effective without troubling dyskinesias of 3.2 hours and a decrease
in the time that oral L dopa was ineffective of 4.1 hours.
[0161] The results indicate that the continuous infusion method
provides increased efficacy in
[0162] Parkinson's patients compared to the discontinuous,
5-deposit/tract method previously used. The reason for this may be
due to an improved distribution of the ProSavin.RTM. vector in the
injected putamen, although it is not possible to assess this in
living patients. Furthermore, the surgical time for vector
administration using the continuous infusion method was almost half
of that compared to the conventional method.
Methods and Materials
[0163] All patients were injected with ProSavin.RTM.
intrastriatally under general anaesthesia using bilateral
stereotaxic injections. A cranial MRI scan was performed prior to
the administration to provide precise injection coordinates for
targeting the sensorimotor putamen region of the putamen.
[0164] For each injection a guide tube of 130 mm in length with a
bore diameter of 1.2 mm was inserted into the correct position
within the brain, using the MRI-derived coordinates, without
entering the putamen. For the conventional method of administration
ProSavin.RTM. was loaded into a Hamilton syringe attached to a 23
gauge point two style bevelled non coring needle, 150 mm in length.
For the continuous infusion method ProSavin.RTM. was loaded into a
Hamilton syringe attached to a 28-gauge needle of the same length.
The needle was lowered into the brain through the guide tube and
penetrated the motor putamen The guide tube was then withdrawn
approximately 10 mm prior to infusion of ProSavin.RTM.. A new guide
tube, Hamilton syringe and needle were used for each hemisphere of
the brain.
[0165] For the conventional method of administration 25 .mu.L of
ProSavin.RTM. was administered to each of five separate tracts in
both brain hemispheres. Each tract received five deposits of 5
.mu.L of ProSavin.RTM.. The deepest deposit was administered first,
the needle was then withdrawn by 1 mm and a second deposit of 5
.mu.L was administered. This was repeated until all five deposits
had been made. Administration was performed manually in each of the
injection tracts at a rate of 1 .mu.L per minute (0.5 .mu.L will be
injected, followed by a 30 second pause before the next 0.5 .mu.L
is injected and so on) until 5 .mu.L was injected into the five
deposits along each tract. The needle was left in situ for one
minute on completion of all five deposits on a single tract.
[0166] For the continuous infusion method ProSavin.RTM. was
administered into three injection tracts per hemisphere. Volumes of
42 .mu.L, 42 .mu.L and 43 .mu.L of ProSavin.RTM. were administered
using continuous infusion at a constant delivery rate of 3
.mu.L/min. The flow rate was controlled by the use of a pump rather
than the manual system described above for the conventional
method.
[0167] All publications mentioned in the above specification are
herein incorporated by reference. Various modifications and
variations of the described methods and system of the invention
will be apparent to those skilled in the art without departing from
the scope and spirit of the invention. Although the invention has
been described in connection with specific preferred embodiments,
it should be understood that the invention as claimed should not be
unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention
which are obvious to those skilled in molecular biology, virology,
neurology or related fields are intended to be within the scope of
the following claims.
* * * * *