U.S. patent application number 09/202564 was filed with the patent office on 2002-06-20 for delivery system for gene therapy to the brain.
Invention is credited to DURING, MATTHEW J., LEONE, PAOLA, SORGI, FRANK L..
Application Number | 20020076394 09/202564 |
Document ID | / |
Family ID | 26722525 |
Filed Date | 2002-06-20 |
United States Patent
Application |
20020076394 |
Kind Code |
A1 |
LEONE, PAOLA ; et
al. |
June 20, 2002 |
DELIVERY SYSTEM FOR GENE THERAPY TO THE BRAIN
Abstract
A gene delivery system which is both safe and results in
long-term expression throughout the brain has been developed. A
lipid-entrapped, polycation-condensed DNA (LPD) system has been
developed for brain gene delivery, using an adeno-associated viral
("AAV") vector in which the transcription unit is flanked by the
145 bp inverted terminal repeats (ITR) of the adeno-associated
virus. This AAV plasmid is more effective than a non-ITR containing
plasmid in vivo . The results show that the LPD-AAV plasmid
complexes efficiently transduce neurons and that gene expression
can persist for over 10 months in the brain. Furthermore, the
intraventricular delivery method with systemic hyperosmolality
results in global gene delivery. The examples show that expression
of the human aspartoacyclase ("ASPA") gene in children with this
metabolic disorder can be obtained over a period of many months to
a year, with functional activity.
Inventors: |
LEONE, PAOLA; (PHILADELPHIA,
PA) ; DURING, MATTHEW J.; (PHILADELPHIA, PA) ;
SORGI, FRANK L.; (SONOMA, CA) |
Correspondence
Address: |
PATREA L. PABST
HOLLAND & KNIGHT, LLP
SUITE 2000, ONE ATLANTIC CENTER
1201 WEST PEACHTREE STREET, N.E.
ATLANTIC
GA
30309-3400
US
|
Family ID: |
26722525 |
Appl. No.: |
09/202564 |
Filed: |
February 8, 1999 |
PCT Filed: |
April 17, 1998 |
PCT NO: |
PCT/US98/07559 |
Current U.S.
Class: |
424/93.2 ;
424/450; 435/320.1; 435/455; 435/458; 435/91.2; 514/44R |
Current CPC
Class: |
A61K 9/1272 20130101;
C12N 15/88 20130101; A61K 47/544 20170801; A61K 48/00 20130101;
C12N 15/86 20130101; C12N 15/87 20130101; C12N 2750/14143 20130101;
A61K 48/0041 20130101; A61K 9/0085 20130101; C12N 9/88
20130101 |
Class at
Publication: |
424/93.2 ;
514/44; 424/450; 435/320.1; 435/91.2; 435/455; 435/458 |
International
Class: |
A61K 048/00; C12P
019/34; A61K 009/127; C12N 015/87 |
Claims
We claim:
1. A method for delivering nucleic acids to cells in the central
nervous system comprising administering lipid-entrapped
polycation-condensed nucleic acid to central nervous system tissue,
wherein the lipid-entrapped polycation-condensed nucleic acid
comprises lipids, polycations, and the nucleic acid to be
delivered.
2. The method of claim 1 wherein the lipids comprise dioleoyl
phosphatidylethanolamine and a cationic lipid.
3. The method of claim 2 wherein the cationic lipid is
3b-[N-(N',N'dimethylaminoethane)carbamoyl]cholesterol.
4. The method of claim 1 wherein the polycations are selected from
the group consisting of polylysine and protamine sulfate.
5. The method of claim 1 wherein the nucleic acid to be delivered
comprises adeno-associated virus inverted terminal repeats flanking
an expression sequence.
6. The method of claim 5 wherein the expression sequence comprises
an early cytomegalus virus promoter, a nucleic acid sequence to be
expressed, and an SV40 poly A signal.
7. The method of claim 6 wherein the nucleic acid sequence to be
expressed comprises a nucleic acid sequence encoding ASPA.
8. The method of claim 1 wherein the central nervous system tissue
is brain tissue.
9. The method of claim 8 wherein the lipid-entrapped
polycation-condensed nucleic acid is administered
intracerebrally.
10. The method of claim 9 wherein the lipid-entrapped
polycation-condensed nucleic acid is administered
intraparenchymally or intraventricularly.
11. A composition for delivering nucleic acids to cells in the
central nervous system comprising lipid-entrapped
polycation-condensed nucleic acid comprising lipids, polycations,
and the nucleic acid to be delivered.
12. The composition of claim 1 wherein the lipids comprise dioleoyl
phosphatidylethanolamine and a cationic lipid.
13. The composition of claim 2 wherein the cationic lipid is
3b-[N-(N',N'dimethylaminoethane) carbamoyl]cholesterol.
14. The composition of claim 1 wherein the polycations are selected
from the group consisting of polylysine and protamine sulfate.
15. The composition of claim 1 wherein the nucleic acid to be
delivered comprises adeno-associated virus inverted terminal
repeats flanking an expression sequence.
16. The composition of claim 5 wherein the expression sequence
comprises an early cytomegalus virus promoter, a nucleic acid
sequence to be expressed, and an SV40 poly A signal.
17. The composition of claim 6 wherein the nucleic acid sequence to
be expressed comprises a nucleic acid sequence encoding ASPA.
18. A construct for expression of a nucleic acid sequence
comprising adeno-associated virus inverted terminal repeats
flanking an expression sequence, wherein the expression sequence
comprises an early cytomegalus virus promoter, a nucleic acid
sequence to be expressed, and an SV40 poly A signal.
19. The construct of claim 18 wherein the nucleic acid sequence to
be expressed comprises a nucleic acid sequence encoding ASPA.
20. A construct for expression of a nucleic acid sequence
comprising adeno-associated virus inverted terminal repeats
flanking an expression sequence, wherein the expression sequence
comprises a nucleic acid sequence encoding ASPA.
21. A method for enhancing delivery of genes to central nervous
system tissue comprising administering compounds which cause
hyperosmolarity in the central nervous system tissue and
administering the genes to the tissue.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention is generally in the area of gene
therapy, and especially prolonged delivery to and expression of
enzymes in the brain.
[0002] Safe and effective methods for gene delivery into CNS
neurons is necessary for successful genetic therapy of neurogenetic
disorders. A large number of both viral and non-viral systems have
been used for gene transfer into neurons both in vitro and in vivo.
The most efficient systems for gene transfer are vectors based on
viruses, most notably Herpes Simplex Virus (HSV) (Geller et al.
1995, During et al. 1994), Adenovirus (Davidson et al. 1993; La Gal
La Salle 1993), Adeno-associated Virus (AAV) (Kaplitt and During,
1995; During and Leone, 1996) and Lentiviruses (Naldini et al.
1996).
[0003] Each of these systems have specific advantages based on
efficiency of transduction, packaging capability, neurotropism and
stability of expression but all share problems with varying degrees
of inflammatory responses, potential for recombination and/or
helper virus contamination and replication competency. These issues
of safety have precluded the use of any these systems for neuronal
gene transfer in humans. Alternative, potentially safer approaches
for brain gene delivery include the use of naked, plasmid DNA as
well as liposome-DNA complexes (Ulrich et al., 1996; Gao and Huang,
1995). Plasmid DNA appears efficient for transducing muscle (Davis
et al., 1993) and liver (following intraportal administration,
Wolf, personal communication). However, direct injection of plasmid
DNA into the brain is very inefficient. Liposome-DNA complexes are
perhaps a little more efficient than naked DNA but compared to
viral vectors remain inefficient. Moreover gene transfer is
generally transient (Roessler and Davidson, 1994; Kozarsdy and
Wilson, 1993).
[0004] The major limitations facing current gene therapy is the
lack of transduction efficiency and the inherent toxicity and
immunogenicity of the most commonly-used vectors. Synthetic
supramolecular complexes may provide for safe and effective gene
delivery vehicles which could be used in the clinic. The specific
problems that gene transfer in the brain present will require both
improvements in the vector system as well as delivery methods for
global gene transfer.
[0005] A major limitation with brain gene transfer relates not only
to the inefficiency of transduction of post-mitotic neurons and
glia but also the inaccessibility of the brain. A problem facing
neurological gene therapy is the need for global delivery and
efficient transduction of cells throughout the brain for most
neurogenetic disorders. Viral vectors do not diffuse well through
brain parenchyma (Doran et al., 1995), perhaps reflecting size and
affinity for cell membrane proteins. A variety of approaches have
been used for global gene delivery. These include multiple
stereotactic injections (Boviatsis et al., 1994); convection
approaches (Oldfield et al., 1994) and hyperosmotic permeation of
the blood-brain-barrier (bbb) (Neuwelt et al., 1994). Each of these
methods is associated with significant morbidity, moreover the
efficacy of each method is limited with bbb permeation effective
for drug and gene deliver to tumors, which already have an impaired
blood brain barrier, but very poor penetration into normal brain
parenchyma.
[0006] It is therefore an object of the present invention to
provide a means for improved delivery and expression of gene
therapy, especially in the brain.
SUMMARY OF THE INVENTION
[0007] A gene delivery system which is both safe and results in
long-term expression has been developed. The method yields
widespread gene delivery throughout the brain. A lipidentrapped,
polycation-condensed DNA (LPD) system has been developed for brain
gene delivery, using an adeno-associated viral ("AAV") vector in
which the transcription unit is flanked by the 145 bp inverted
terminal repeats (ITR) of the adeno-associated virus. This AAV
plasmid is more effective than a non-ITR containing plasmid in
vivo. In addition, an intraventricular delivery system has been
developed to obtain widespread, global delivery.
[0008] The results show that the LPD-AAV plasmid complexes
efficiently transduce neurons and that gene expression can persist
for over 10 months in the rat brain. Furthermore, the
intraventricular delivery method with systemic hyperosmolality
results in global gene delivery. The examples show that expression
of the human aspartoacyclase ("ASPA") gene can be obtained over a
prolonged period of time. Trials in children with this metabolic
disorder show that gene expression can be obtained over a period of
many months to a year, with functional activity.
DETAILED DESCRIPTION OF THE INVENTION
[0009] A gene delivery system which is both safe and results in
long-term expression has been developed. Furthermore, a method to
obtain widespread gene delivery throughout the brain has been
developed.
[0010] I. The Delivery System
[0011] Lipid Carriers
[0012] A lipid-entrapped, polycation-condensed DNA (LPD) system for
brain gene delivery has been developed. The LPD particles are
small, about 60-80 nm, monodispersed and colloidally stable (Gao
and Huang, 1996). These particles have been shown in vitro to be
highly effective in transduction efficiency (approximating that of
adenoviral vectors) and safe (Huang, 1997).
[0013] The LPD complexes represent a significant step forward in
non-viral, synthetic delivery systems:
[0014] 1) The CD-Chol/DOPE liposome backbone is safe and has been
used in 2 published clinical trials (Kaplen et al., 1995; Nabel et
al., 1993) and 3 other ongoing protocols.
[0015] 2) Both poly-L-lysine and protamine have been used
clinically and appear safe with protamine being widely used as a
heparin antagonist clinically.
[0016] 3) AAV plasmids have previously been shown to enhance
transduction efficiency and stability (Vieweg et al., 1995; Philip
et al., 1994).
[0017] 4) Both protamine and poly-l-lysine have nuclear
localization signals which may also improve transduction
efficiencies.
[0018] 5) Both protamine and poly-l-lysine efficiently complex
plasmid DNA and result in condensation of the DNA. This condensed
DNA changes from an external diameter of approximately 200 nm to a
50-100 nm size when further complexed with the cationic liposome.
These nano-LPD particles efficiently transduce post-mitotic cells
including neurons and glia.
[0019] Other carriers such as liposomes, polycationic complexes and
polymeric carriers can also be used, but are not preferred.
[0020] AAV Vectors
[0021] The gene to be delivered is placed into an adeno-associated
viral vector. In the preferred embodiment, the transcription unit
is flanked by the 145 bp inverted terminal repeats (ITR) of the
adeno-associated virus. These ITRs have been shown to both enhance
and prolong gene expression in both prostatic and lympocytic cell
lines in vitro (Vieweg J, et al., 1995; Philip R, et al, 1994). The
vector also includes a CMV promoter. This AAV plasmid is more
effective than a non-ITR containing plasmid in vivo.
[0022] Other viral vectors can also be used, including retroviral
vectors and adenoviral vectors, although these are not preferred
for regulatory issues.
[0023] Means for Administration
[0024] In the preferred application for this technology, the viral
vector is administered to the brain using a syringe or catheter.
Intraventricular delivery is used to obtain widespread, global
delivery.
[0025] Uptake is enhanced using agents such as mannitol, which
cause a systemic hyperosmolality. For example, intraparenchymal
penetration is enhanced by lowering brain interstitial pressure by
the use of systemic mannitol (Rosenberg G A, et al., 1980).
[0026] II. EXAMPLES
[0027] The results show that the LPD-AAV plasmid complexes
efficiently transduce neurons and that gene expression can persist
for over 10 months in the rat and human brain. The intraventricular
delivery method with systemic hyperosmolality results in global
gene delivery. The LPD complexes also result in long-term
expression and following intraventricular hyperosmolar
administration can lead to global gene expression. LPD complexes
provide for the excellent efficacy/toxicity ratio.
[0028] The examples show that expression of the human
aspartoacyclase ("ASPA") gene can be obtained over a prolonged
period of time. A deficiency in ASPA cause Canavan disease, or
spongy degeneration of the brain. The human ASPA gene has been
cloned, as described by Kaul, et al., Genomics 21(2), 364-370
(1994). Three point mutations predominantly found in Ashkenazi Jews
cause the expressed enzyme to be completely inactive. Kaul, et al.,
Am. J. Hum. Genet. 55(1), 34-41 (1994). Other mutations have been
found in non-Jewish patients. Kaul, et al., Am. J. Hum. Genet.
59(1), 95-102 (1996).
[0029] Children who are deficient in this enzyme quickly loose
brain function, deteriorating to a vegetative state by a few years
of age, and dying shortly thereafter. There is no treatment for
these children.
Example 1
Construction of the AAV Vector
[0030] Materials and Methods
[0031] The recombinant DNA used for the study contained three
general elements. The plasmid backbone is derived from pEMBL and is
a high copy E. coli based plasmid with an ampicillin resistance
gene and an E. Coli origin of replication. The plasmid contains 145
base terminal repeats from the human non-pathogenic, parvovirus
adeno-associated virus (AAV) (Kaplitt and During, 1995). These
sequences are non-coding and form a hairpin secondary structure
which is important for both transduction efficiency of the plasmid
as well as stability of expression of the flanked transcription
unit (Philip et al., 1994; Viewig et al., 1995). The third
component of the plasmid which is spanned by the AAV terminal
repeats is the transcription unit consisting of the CMV promoter
(650 bases), and either the E. Coli lacZ gene (encoding the marker
protein, .beta.-galactosidase, AAVlac) or the full-length human
ASPA cDNA (1.4 kb, obtained from Drs. Kaul and Matalon of Miami
Children's Hospital, pAAVaspa) and the SV40 early polyadenylation
signal (400 bases).
[0032] Regulatory Elements:
[0033] The promoter is the 650 base pair early cytomegalus virus
(CMV) promoter which was obtained from the commercial (Invitrogen,
Inc.) plasmid pcDNA1. This promoter is a very strong constitutive
promoter active in all eukaryotic cell types. There has been some
variable loss of expression associated with the use of this
promoter in studies of gene transfer which may reflect methylation.
However, this promoter has been widely used in clinical gene
transfer experiments. No loss of expression over time has been
observed in lentiviral vectors in the brain (Naldini et al., 1996)
and AAV vectors in muscle (Xiao et al., 1996). The polyadenylation
signal was the XV40 polyA derived from the Invitrogen plasmid
pREP4.
[0034] Steps Used to Derive the DNA Construct:
[0035] Plasmid pSub201 (Samulski et al., 1989) was digested with
XbaI to remove nearly the entire AAV genome, leaving only the
terminal repeats. A CMV promoter-lacZ gene--SV40 polyA signal
cassette was isolated from plasmid pHCL (Kaplitt et al., 1994) by
digestion with SpeI and XbaI, and this was inserted into
XbaI-digested pSub201 to create pAAVlac. A second plasmid was
created (pAAV-CMV-polyA) by digestion of pAAVlac with HindIII and
XbaI to remove the lacZ gene and polyA signal, followed by
insertion of a HindIII-XbaI fragment from pREP4 (Invitrogen),
containing a polylinker and SV40 polyA signal. This plasmid was
digested with NheI and NotI, followed by insertion of a human ASPA
cDNA released from the plasmid pGCMV-ASPA (Kaul et al., 1993) by a
XbaI--NotI digestion. The sequence of the final construct was
confirmed by automatic sequencing on an ABI Model 377 sequencer.
The plasmid was made under sterile conditions in the presence of
ampicillin using the E. Coli strain DH5.alpha. and purified in
facilities at the University of Pittsburgh.
Example 2
Delivery to and Expression in Animals of .beta.-Galactasidase.
[0036] Materials and Methods
[0037] Liposomes and Polycations
[0038] The liposome was made under sterile, good manufacturing
processing ("GMP") conditions. The liposome is a mixture of the
neutral lipid DOPE (dioleoyl phosphatidylethanolamine) with a
cationic liposome, DC-Chol
(3b-[N-(N',N'dimethylaminoethane)carbamoyl]cholesterol). The
polycations used included poly-L-lysine (18,900 m.w., Sigma, St.
Louis) and protamine sulfate (USP grade, Eli Lilly).
[0039] Evaluation of Gene Expression
[0040] X-gal Histochemistry
[0041] Animals were deeply anesthetized and transcardially perfused
with PBS followed by 2% paraformaldehyde with 5 mM EGTA. This
fixation method completely eliminates staining due to endogenous
enzymes (Kaplitt et al., 1991; Kaplitt et al., 1994). Brain
sections were stained with the histochemical substrate X-gal as
previously described (During et al., 1994; Kaplitt et al.,
1994).
[0042] NF, GFAP and B-galactosidase Immunocytochemistry
[0043] For immunocytochemical (ICC) analysis of brain sections,
rats were deeply anesthetized with chloral hydrate and perfused
with 1 M PBS (ph 7.3) followed by 4% paraformaldehyde (PF). Brains
were removed and post-fixed (3-4 hours) in 4% PF followed by
ascending sucrose solutions (10/15/30% in PBS). Sections (7-30
.mu.m) were cut in a cyrostat (Reichert-Jung) and mounted on
polylysine-coated slides. Sections were initially incubated in
blocking buffer (5% goat serum (GS)15% normal horse serum (NHS) in
1 M phosphate buffered saline (PBS)), followed by 2-4 hours
incubation at room temperature with mouse Ig antibody.
[0044] Intracerebral Gene Delivery
[0045] Groups of rats were maintained in a housing facility at the
University of Auckland School of Medicine under controlled lighting
(12 hour day/light cycle), temperature and humidity with ad libitum
access to food. The animal protocol was approved by the Yale Animal
Care and Use Committee and the University of Auckland Animal Ethics
Committee. All surgeries were carried out under general anesthesia
using ketamine/xylazine (70/7 mg kg.sup.-1) administered
intraperitoneally. A stereotactic frame (Kopf) was used for all
surgeries:
[0046] Intraparenchymal injections were made into the hippocampus
(A-P=-2.9; M-L=1.6; D-V=2.0), striatum (A-P=0.6; M-L=2.4; D-V=5.0)
or intracerebroventricularly A-P=0.9; M-L=1.5; D--V=3.2).
[0047] The following groups of animals were studied:
[0048] 1) Intraparenchymal LPD (poly-L-lysine)
[0049] 2) Intraparenchymal lipofectamine
[0050] 3) Intraparenchymal LPD (protamine)
[0051] 4) Intraparenchymal LPD (plasmid without AAV ITR)
[0052] 5) Intraventricular LPD (poly-L-lysine)
[0053] 6) Intraventricular LPD (protamine)
[0054] 7) Intraventricular LPD (protamine plus systemic
mannitol)
[0055] 8) Intraventricular LPD (AAVaspa; poly-L-lysine with
systemic mannitol)
[0056] 9) Repeated dosing of LPD
[0057] Results
[0058] Intraparenchymal delivery of an AAV expression plasmid
together with lipofectamine resulted in limited expression
surrounding the needle-tract with little penetration into the
tissue. Furthermore, expression diminished over time although a few
positive cells were apparent at one month following surgery. In
comparison, the delivery of the same plasmid following condensation
with poly-L-lysine and complexing the CD-Chol/DOPE resulted in a
marked increase in expression extending to ne month. The use of
protamine instead of poly-L-lysine resulted in a further
enhancement of expression at both 1 week and one month. With a
volume of 2-4 microliters which resulted in local needle tract
expression with lipofectamine, more widespread expression was
obtained with the LPD complex. Furthermore, if the injection volume
was increased to 8 microliters much wider expression was obtained
with preferential spread of the complex and gene expression along
white matter tracts and blood vessels.
[0059] The use of the AAV ITR-containing plasmids resulted in
similar expression at 1 week but greater expression at one month
and 8 months when compared to the LPD complex using the identical
expression cassette not flanked with the ITRs.
[0060] Intraventricular administration of the LPD complexes
resulted in expression in periventricular cells extending into
parenchyma. The parenchymal spread was enhanced by the use of
systemic mannitol which drops interstitial pressure and increases
CSF flow away from the ventricles.
[0061] In the rat, the LPD/AAVlac complex has been injected into
both brain parenchyma as well as intraventricularly. Results have
shown that in the first week (Days 3 to 7) following the injection
of 2 to 6 microliters of the complex at a concentration of 300
.mu.g DNA per ml of LPD, 10.sup.3 cells are transduced down the
needle track and in the vicinity of the LPD diffusion (the
LPDlplasmid complex) which follows white matter tracks within the
brain and tracks along planes between brain regions and groups of
cell nuclei. At two and six months, 10.sup.2-10.sup.3 remain
readily detectably with X-gal staining, suggesting minimal loss of
expression over this time period.
Example 3
Expression of Functional ASPA in Human Cells.
[0062] In vitro studies used the 293 human embryonic kidney cell
line. As there is not an ASPA antibody suitable for
immunocytochemical or immunoblot analysis, both in vitro and in
vivo efficacy were initially assessed using LPD with a marker gene
(E. coli lacZ plasmid expressing .beta.-galactosidase).
[0063] In vitro efficacy is dependent on the relative
concentrations of DNA, liposome and plasmid and under optimal
concentration efficiencies of 80% are obtained. In vivo efficacy
using injection volumes of 2-50 microliters (at a DNA concentration
of 300 .mu.g/ml of LPD) demonstrate transduction of
10.sup.3-10.sup.5 cells.
[0064] Cultured cell results show mean levels of ASPA enzyme
activity of 0.40.+-.0.12 mU/mg protein following LPD/ASPA
transduction of 293 human embryonic kidney cells with an
approximate 5% transduction efficacy. This compares to activity of
1.04.+-.0.04 mU/mg protein in rat cortex. If one percent of enzyme
activity is sufficient to influence disease progression, then one
would aim for a transduction efficiency of approximately 0.1% in
defined (periventricular) regions.
Example 4
Safety and Efficacy Studies in rats.
[0065] The same techniques described in example 2 were used to
administer the vector of example 1 to rats and to monkeys.
[0066] Expression out to 10.5 months using the LPD/AAVaspa
construct following intraventricular administration has been
observed. As there is no antibody to ASPA suitable for
immunohistochemical analysis, one can not distinguish at the
protein level the transgene-encoded ASPA from the wild enzyme.
RT-PCR was therefore used to follow ASPA transgene expression.
Widespread expression was obtained throughout the brain, extending
to 10.5 months following intraventricular administration. This
supports the expression data on B-galactosidase expression
following intraventricular of LPD/AAVlac. Using an intraventricular
approach together with systemic mannitol to reduce interstitial
pressure, widespread expression extending several millimeters from
the ventricular surface was observed.
[0067] Although the number of transduced cells as a percentage of
total brain cells (target cells) is low, it can reach 106 cells
following high volume (10-20 microliters) intraventricular
injection. From the in vitro work, it is known that relatively low
transduction efficiencies can lead to wild-type levels of the
enzyme and that the protein is functionally active.
[0068] Neurosurgical stereotactic injections were performed on a
total of 400 rats with either the AAVaspa or AAVlac plasmid,
DC-Chol/DOPE and protamine (LPD) complex and PBS-treated controls
without any toxicity or adverse effects on rat behavior, feeing or
general health. All these rats were weighed on a twice weekly basis
and were observed for coat quality (grooming behavior) and
conjunctivitis (probably the most sensitive indicator of general
stress) as well as feeding and drinking behavior. A summary of
animal studies follow:
1 Experimental Route of Duration of Animals and # Administration
Volume Study Gene Outcome Rats, n = 3 .times. 60 groups (4 i.c.v.
5, 10, 20 7 days, 1, 3, 6 LacZ or No toxicity volumes by 5
timepoints by 3 50 .mu.l and 9 months ASPA or treatments) PBS Rats,
n = 3 .times. 60 groups (4 i.p 2, 5, 8, 3, 7 days; 2, 6, LacZ or No
toxicity volumes and 5 timepoints) 12 .mu.l 8 months ASPA or PBS
Rats, n = 5 .times. 3 treatment i.c.v. repeated dose at 50 .mu.l 3
months LacZ or No toxicity groups 1 month ASPA or PBS Rats, n = 5
.times. 3 treatment i.p. repeated does at 1 12 .mu.l.mu.l 3 months
LacZ or No toxicity groups month ASPA or PBS Rats, n = 2 for 6
groups (2 i.c.v. very high dose 100, 250 .mu.l 2 weeks LacZ or Well
tolerated* doses and 3 treatments) ASPA or PBS i.c.v. =
intracerebroventricular i.p. = intraparenchymal
[0069] All rats in this high dose group (including control rats
injected with the reporter gene and sterile Phosphate Buffer
Saline) displayed sleepiness and hypomotility (measured by the
open-field test and grooming behavior) during the 24 hours
following the surgery. Rectal temperature raised up by 2.degree. C.
in all animals for 10 hours post-surgery. The feeding rate, which
decreased mildly post surgery in all animals, went back to baseline
after 48 hours.
[0070] An additional subset of five animals were infused with
AAVaspa/LPD (50 microliters) via an indwelling ventricular cannula.
These animals were followed with daily weights, temperature and
behavioral tests. The animals were followed with rectal
temperatures both in the morning and evening.
[0071] Behavioral analysis included:
[0072] 1) Open filed behavior and locomotion;
[0073] 2) Spatial navigation behavior using the Banes circular
platform;
[0074] 3) Response to novel environment;
[0075] 4) Rigidity/catatonia testing;
[0076] 5) Motor coordination and climbing using the inclined beam
test;
[0077] No adverse effects were noted over a one month period.
Histological analysis following sacrifice. Animals were euthanized
and brains perfused with paraformaldehyde. Brains were sections at
30 micron thickness. Sections were stained with H&E and
underwent microscopic evaluation. No inflammatory
responses/neuronal injury.
[0078] Although light microscopy evaluation of H & E stained
tissue did not reveal any inflammatory cell infiltrate into the
brain and normal neuronal and glial morphology with complete
integrity of the tissue, a recent study by Byrnes et al. (1995) has
shown that adenoviral vectors result in inflammation and increased
expression of immunological markers including MHC Class I
expression and T cell infiltration. A group of 6 rats were
therefore injected with the AAVaspa/LPD complex intraventricularly
(20 .mu.l) and a group of 6 rates intraparenchymally (8 .mu.l) to
determine whether the LPD complex resulted in T cell infiltration
and a change in MHC expression. A control, saline (PBS) treated
group of 6 animals was used as a control for both groups. Animals
were sacrificed at 4 days and 1 month with brains perfused and
fixed. Forty micron coronal sections were cut on a cryostat and the
primary antibodies OX-18 (purified antibody which reacts with a
non-polymorphic determinant of rat MHC class I antigen) and OX-35
(reacts specifically with the CD4 antigen on T helper cells,
thymocytes, and activated macrophages and microglia) were applied
(both obtained from Pharmacia). In none of the
intraventricular-treated animals were CD4-IR cells identified nor
was there any neuronal MHC Class I expression. In the
intraparenchymally-injected animals which were sacrified at 4 days,
a small number of CD4iIR cells as well as OX-18 staining was
present in the immediate vicinity of the needle tract, however this
increase was noted in both PBS and LPD-injected animals. At one
month, there was no intraparenchymal staining with either OX-18 or
OX-35 in either group of animals. Although studies did not include
an adenoviral vector-treated group, in comparison with the data
presented by Charlton for adenovirus (Byrnes et al., 1995) and HSV
(Wood et al., 1994) in which the vectors resulted in severe
widespread expression of MHCI antigents, or LPD and PBS-treated
animals had significantly less staining of these markers with only
moderate staining immediately around the injection site at 4 days
with OX-18 and few CD4 positive cells with complete resolution of
the inflammation at one month. Moreover, the intraventricular
injection did not result in any parenchymal inflammation. Together
with the persistent expression at 10 months (in contrast to
adenoviral vectors with marked loss of expression over 1-3 months),
this data suggests that the LPD complexes are significantly less
immunogenic than current generation viral vectors.
[0079] PCR analysis of testes was carried out in 10 LPD-treated
rats and a group of 15 cynomologous monkeys treated with other
vectors (intraparenchymal and intraventricular injections of
recombinant AAV and AAV plasmids with poly-L-lysine/liposome
complexes). Foreign DNA was not detected in the gonads. In
addition, a PCR signal was not detected in other non-CNS tissues
including liver, spleen and kidney.
Example 5
Expression in initial Human Clinical Trials
[0080] Approval for a Phase I clinical protocol was obtained from
the following committees: Yale University School of Medicine Human
Investigation Committee; North Health (Auckland Hospital, New
Zealand) Ethics Committee; University of Auckland Ethics Committee
Biosafety Committee (Auckland Hospital); and New Zealand Gene
Technology Advisory Committee.
[0081] Two children (L.K. aged 19 months and A.M. aged 24 months)
were enrolled in the initial Phase I trial using the LPD/ASPA
complex.
[0082] Clinical Report Summary at 6 Months
[0083] The surgical procedure was uncomplicated apart from a mild,
transient post-operative fever (<40.degree. C.) in both children
lasting for less than 72 hours. One child (A.M.) had some minor
pulsatility over the burrhole site associated with facial dependent
oedema which settled over 72-96 hours with complete resolute of the
presumed dural CSF leak. CT Scans post surgery were completely
normal. Both children were drowsy for the first 24-48 hours
post-surgery but by 3 days were as alert (or more so) than
baseline. Blood tests, both hematological and biochemical, remained
within the normal range. Both children were discharged at one week
following surgery. Findings are reported for evaluations prior to
surgery and 1, 2, 4 and 6 months postoperatively.
[0084] Prior to surgery, L.K. was evaluated using the
Gesell-Provence Developmental Schedules (GPDS), a
neurodevelopmental instrument that provides a developmental age
equivalent based on a profile of functioning across five
domains--gross and the fine motor, adaptive or problem solving,
language and social development. The severity of L.K.'s
developmental delays precluded an accurate age equivalent or
developmental quotient as she demonstrated considerable variation
in her performance across developmental domains and a basal
performance level could not be obtained. However with this caveat
in mind, her highest level of functioning was less than a 12 week
age equivalent and in some areas less than an 8 week equivalent.
Notably L.K. needed complete head support, arched her back in
extension when moved, did not reach for objects, and had minimal
grasp and extremely limited visual tracking. At one month post
surgery, L.K. was re-evaluated by the same observer. The most
notable difference was overall greater awareness and alertness. She
was more responsive and smiled more and often spontaneously. She
also initiated social interaction on her own. Facial expressions
were brighter and more differentiated. She was able to chew and
swallow solid foods in a coordinated fashion. She was able to hold
her head unsupported for a few seconds and was able to turn from
her side to her back and bear some weight on her legs. L.K. also
had notable gains in language and social skills. She was beginning
to make a number of differentiated vocalizations including
consonant sounds. She responded to simple statements like "I want a
drink" by opening her mouth and used an open mouth to indicate
"more". Her vocalizations appeared to indicate different needs and
she appeared to understand and respond to a number of phrases (by
head turning, smiling, visual regard). Her development age
equivalent appeared to be at 3 months. At six months following
surgery, further skills were achieved. K.K. was able to hold her
head up unsupported for up to 8 minutes. She was able to grasp
objects, and was much clearer in expressing her wants and
displeasure.
[0085] A.M. was evaluated at age 19 months (5 months prior to
surgery) using the GPDS, at which time she had notable head lag and
was unable to hold her head unsupported. She was not reaching for
objects but was able to hold an object briefly if placed in her
hands. She appeared interested in novelty and turned to sound. She
laughed and cooed but made no consonant sounds. Her developmental
age was between 12-16 weeks. At reassessment immediately prior to
surgery, A.M. had lost a number of developmental skills including
less responsivity to environmental stimuli, poorer head control and
much increased muscle tone with marked overall rigidity. Her
baseline developmental age equivalent immediately presurgery had
deteriorated to less than 12 weeks. At one month following surgery,
A.M. was more interested in and aware of her surroundings, had more
differentiated facial expressions, had better visual tracking and
was making eye contact when spoken to. She was turning to look and
moved her head back and forth. She also had less drooling, more
coordinated swallowing and regular bowel motions. A.M. was
significantly less rigid with more spontaneous and purposeful
movement. She was able to put her arms around her parents neck when
held, and also showed some motor activity in the midline evidenced
by her grasping hands together on several occasions. Her
developmental age equivalent at that time was approximately between
12-16 weeks. At 2 months post surgery, A.M. had maintained her
developmental state with some minor advances. Specifically she
showed greater interest and awareness of her surroundings, more
differentiated facial affect and greater social interactiveness.
She was also better at chewing and swallowing. However, her
neurological state subsequently plateaued with no significant
changes between 2 and 6 months post surgery.
[0086] In addition to developmental and neurological assessment,
both children underwent repeated visual evoked potentials (VEDPs)
and proton spectroscopy of regional brain NAA. L.K. is baseline VEP
to pattern reversal was performed binocularly, since she would not
tolerate monocular testing. There was significant bilateral delay
with the P100 (Positive wave observed in normals at 100 ms) latency
to 154.4 ms. Moreover the amplitude from P1 to N2 was 0.83 uV
(Normal >1.5 uV). This test suggested bilateral optic pathway
pathology. On retesting at 2 months post surgery, VEPs were normal
with latency of 85.8ms and amplitude of 2.39 uV in both eyes. At 5
months post surgery, VEPs remained within the normal range. L.K.
was also assessed by an ophthalmic surgeon in view of hyperopia and
astigmatism. Prior to surgery, significant deterioration had
occurred with loss of fixation, amblyopia, exotropia, horizontal
and occlusion nystagmus. On re-evaluation, 2 months post surgery,
L.K. showed good fixation, no signs of amblyopia, partial
resolution of exotropia and the nystagmus had completely resolved.
A.M. was unable to cooperate sufficiently to obtain interpretable
VEPs both prior to, and following surgery.
[0087] Baseline NMRS was performed at Yale, however a system
upgrade required that postoperative NMRS was conducted at Childrens
Hospital in Philadelphia (CHOP) and an additional control group of
5 Canavan children (untreated and ages 6-30 months) was also
studied at CHOP.
[0088] L.K.'s NAA baseline concentration was quantiated at 15.4 mM
in the occipital region. Parietal and frontal lobe concentrations
were also significantly above control values despite an
occipito-frontal gradient with lower values anteriorly on proton
spectroscopic imaging. Mean concentration and standard deviation
(S.D.) of NAA from 8 control spectra (in 8 subjects) measured from
frontal, parietal and occipital lobe is 9.8.+-.0.6 mM (no
significant regional differences). At one month post surgery,
occipital region remained elevated above normal at 14.5 mM, however
both parietal and frontal lobes were now within the low normal
range, NAA=9.4 and 8.4 mM respectively. At 4 months the occipital,
parietal and frontal NAA concentrations were 16.2, 10.0 and 9.1 mM
respectively. Therefore normal levels of NAA were maintained in
both parietal and frontal regions with high levels persistent in
the occipital lobe.
[0089] A.M.'s baseline NAA concentration was 15.6 mM in occipital
lobe. At one month NMRS at CHOP of occipital, parietal and frontal
regions showed concentrations of NAA of 12.8, 10.0, 9.6 mM. At 4
months, levels were 12.7, 12.6 and 12.9 mM respectively.
[0090] Untreated Canavan children scanned at CHOP using the same
magnet and parameters gave mean +S.D. values of 15.7+1.5, 13.9+2.0,
13.6+1.8 mM for occipital, parietal and frontal lobes
respectively.
[0091] These NMRS results show normal concentrations of NAA in both
frontal and parietal regions for both children at one month, with
maintenance of normal values in these regions at 4 months for L.K.,
but a reversion to the Canavan range at this timepoint for A.M.,
although A.M.'s occipital NAA concentration of 12.7 mM remain lower
than all other Canavan children (range 14.7-18.0 mM).
[0092] Clinical Report Summary at 12 Months
[0093] Report on L.K.
[0094] Biochemical
[0095] Mean concentration and standard deviation (S.D.) of NAA from
8 control spectra (in 8 subjects) measured from frontal, parietal
and occipital lobe was 9.8+0.6 mM (no significant regional
differences). L.K.'s baseline NAA levels (carried out at Yale) was
elevated in all brain regions (15.4 mM in occipital cortex with a
posterior-anterior gradient, but frontal lobe levels markedly
elevated above control values). At one month post surgery,
occipital region NAA remained elevated above normal at 14.5 mM,
however both parietal and frontal lobes were within the low normal
range, NAA=9.4 and 8.4 mM respectively.
[0096] At 4 months the occipital, parietal and frontal NAA
concentrations were 16.2, 10.0, and 9.1 mM respectively and at 9
months were 16.5, 10.2 and 10.4 mM respectively. A proton NMRS
spectroscopy scan was conducted at 12 months following surgery in
late February, 1997. The report shows that her NAA levels in the
occipital lobe was 16.9 mM, parietal lobe 10.4 mM and the frontal
cortex 9.8 mM. These values are essentially unchanged from the
values obtained at 9 months and both frontal and parietal lobes
remain within the normal range for age-matched children.
[0097] MRI
[0098] Serial MRI studies of the brain were performed using a 1.5
tesla magnet conducted at the same time as the proton NMRS.
Unenhanced T1-weighted images were acquired in the sagittal and
transverse planes. The baseline study (within 3 weeks of surgery)
showed diffuse, abnormal prolongation of the T1 signal within the
subcortical and deep white matter. These multiplanar images with
increased T1 relaxation time in white matter are consistent with
the spongiform degeneration of oligodendrocytes characteristic of
Canavan Disease. At four months post surgery a repeat examination
was performed and compared with the previous study. Again, diffuse,
abnormal prolongation of the T1 signal within the subcortical and
deep white matter was demonstrated which was unchanged from the
initial study. At 9 months, the T1-weighted sequences showed less
pronounced hypointensity than the previous studies. In particular,
the subcortical white matter showed a decrease in the abnormal
prolongation of the T1 signal. At 12 months post surgery, a myelin
quantitation MRI study was repeated using sagittal T1-weighted,
axial T1 and T2, axial FLAIR and coronal T2. The exam was compared
to previous studies. At this time, it was noted that there has been
definite interval myelination. Specifically, there was evidence of
improved (although not normal) myelination of the corpus callosum
as well as the basal ganglia and the posterior limb of the internal
capsule. The myelination pattern at this latest time is roughly
what would be expected in a 4 month old infant. The myelination in
the baseline and 3 month studies was more suggestive of a pattern
expected nearer birth. This evaluation and interpretation was
conducted by Dr. Larissa Bilianuk, M.D. and Dr. Robert Zimmerman,
board certified neuroradiologists at Childrens Hospital of
Philadelphia. These changes in the MRI are suggestive of a more
developed myelin pattern i.e., remyelination and/or reduced
spongiform degeneration of the oligodendrocytes. In addition, the
latest proton NRMS data suggests that some level of transgene
expression has persisted out to 12 months in L.K. Furthermore, the
MRI data suggesting new myelin formation is dramatic, however it is
consistent with, and perhaps to be expected, in light of the
observed gain in neurological function (being able to hold her head
up) and normalization of visual evoked potentials. Moreover, the
timing of reaching the visible threshold for detecting a change in
the abnormal T1 relaxation time is consistent with our estimates
for new myelin formation following biochemical normalization.
[0099] Neuro-Ophthalmic Evaluation
[0100] In the months prior to surgery, significant deterioration
had occurred with loss of fixation, amblyopia, exotropia,
horizontal and occlusion nystagmus. On re-evaluation, 2 months, 6
months, 9 months and again at 12 months post surgery, L.K. showed
good fixation, no signs of amblyopia, partial resolution of
exotropia and the nystagmus had completely resolved.
[0101] Visual Evoked Potentials
[0102] The visual evoked potentials had markedly delayed latency
and reduced amplitude prior to the surgical intervention. On repeat
testing at 2 months post surgery, the VEPs lay within the normal
range. At the most recent assessment (Mar. 12, 1997) VEPs as well
as AEPs (auditory evoked potentials) were normal.
[0103] Neurodevelopmental Assessment
[0104] Neurodevelopmental assessments have shown continual further
improvements in L.K. She has been re-evaluated at 12 months post
surgery (chronological age 33 months) and her functional level in
scattered skill development in specific areas of cognition,
expressive language and social emotion is the equivalent of a
normal 18 to 24 months age child. L.K.'s speech therapist has
determined that she is able to understand and responds
appropriately and selectively to simple commands in English and
Spanish (her nanny's native language is Spanish). There were other
the very evident improvements in L.K.'s neurological state. She was
far more interactive with improved visual regard and motor function
reaching out with both hands, in addition to holding her head up
for extended periods.
[0105] Report on A.M.
[0106] Biochemical
[0107] Proton MR spectroscopy examinations were performed on
Magneto S using single vowel STEAM. At one month post surgery, MRS
at CHOP of occipital, parietal and frontal regions showed
concentrations of NA of 16.6 mM, 10.1 mM and 9.9 respectively. At 4
months post surgery, levels were 11.6 mM, 11.5 mM and 12 mM
respectively. At 6 months post surgery, levels were 11.7 mM, 11.7
mM and 11.8 mM. At 9 months post surgery, levels were 12.7 mM, 13.8
mM and 12.5 mM. At one month, both parietal and frontal lobe NA
levels were within the normal range. However, by 4 months these
values were elevated and have remained elevated since. Only A.M's
occipital NA concentration of 12.7 mM remains lower than the
Canavan range. Indeed untreated Canavan children scanned at CHOP
using the same magnet and parameters gave mean +S.D. values of
15.7+1.5, 13.9+2.0, 13.6+1.8 mM for occipital, parietal and frontal
lobes respectively.
[0108] MRI
[0109] A.M.'s MRI of the brain was performed with the same magnet
and same parameters of L.K. as MRI. The 9 month MRI report
demonstrates diffuse prolongation of the T1 and T2 signal in the
cerebellar and cerebral white matter. This is slightly more marked
around the atria and occipital horns of the lateral ventricles.
Abnormal signal is seen within the globus pallidi as well as the
thalami. There is abnormal signal within the discending tracts of
the brainstem, including dorsal midbrain and pons as well as the
cerebral peduncles. There is dilatation of the atria and occipital
horns of the lateral ventricles, likely on the basis of ex vacuo
change. No deterioration had been noticed since surgery, but unlike
L.K. there was no significant changes in the white matter signal.
A.M.'s 12 month follow-up MRI report shows no interval
remyelination.
[0110] Neurological Examination
[0111] A.M. was increasingly rigid prior to surgery showing
evidence of neurological deterioration. Following gene transfer,
she was re-evaluated at one month. Significant improvements were
noted in A.M. and she was assessed as a 12-16 week age equivalent
(8-12 week prior to surgery). At six months, there was a plateauing
in the clinical state of A.M. but she remained at a higher level of
neurological function than that prior to surgery. The improvement
in neurological and developmental state was consistent with the
transient normalization of brain NA in defined brain regions. A.M.
had smaller and relatively short-lived changes than L.K. with
respect to NA perhaps reflecting a transient CSF leak following
surgery and is likely therefore to have had less CNS gene transfer.
This is also consistent with a lesser clinical improvement than
that observed with L.K. No significant changes have been determined
in her 12 month follow-up examination, with her clinical state
essentially stable over the last 9 months.
[0112] Neuropthalmic Evaluation
[0113] Over the 12 month period following surgery there have been
no signs of amblyopia, exotropia or nystagmus.
[0114] Visual Evoked Potentials
[0115] A.M. was unable to cooperate sufficiently to obtain
interpretable VEPs both prior to, and following surgery at 3
months. At 12 months, A.M. was able to cooperate sufficiently. At
this time her pattern reversal VEP shows a bifid cortical potential
which was reproducibly demonstrated. This bifid response may
represent an abnormality, although the latencies of the second
negative and positive deflections are within the normal range. The
significance of the bifid pattern can not be resolved in a child of
this age and would require lower field stimulation alone.
[0116] Neurodevelopmental Assessment
[0117] A.M. current developmental age equivalent in all areas of
functioning is 12-16 weeks (compared to 8-12 weeks). However, she
showed some notable changes since the gene therapy procedure which
stabilized at 6 months post-surgery. The changes include an
increase in her interest and awareness of her surroundings, more
eye contact with visual tracking, more differentiated facial
expression, greater social interactiveness and more coordinated
bowel function and swallowing with less drooling. No further
improvements have been noticed since the neurological assessment at
6 months post-surgery. In summary A.M.'s improvements have not been
as marked as in L.K., although she also has maintained her
improvement with some slight gains, for example, she rolled over
and fell out of bed last month. Although this sounds trite, Canavan
children do not fall out of bed, they are virtually immobile,
mostly rigid with very few spontaneous movements and unable to roll
over. The improved motor function in A.M. is also matched by
improved responsiveness, although her level of functioning is not
as high as L.K.
[0118] Clinical Outcome Summary
[0119] At twelve months following surgery, both children are at a
higher level of neurological function than that prior to surgery.
This study as a Phase I trial with only 2 children was not designed
for and does not have statistical power to assess clinical
efficacy. Importantly however, it does demonstrate safety and
tolerability in these two children with no significant adverse
effects and only a transient postoperative fever. The improvement
in neurological and developmental state is consistent with a
normalization of brain NA in defined brain regions. Moreover, A.M.
had smaller and more transient changes with respect to NA perhaps
reflecting the transient CSF leak following surgery and is likely
therefore to have had less CNS gene transfer. This is also
consistent with a lesser clinical improvement than that observed
with L.K. Although lowering of CSF pressure may result in transient
benefit in Canavan children with raised intracranial pressure
(personal communication, S. Gluckman), both children studied in
this trial had normal opening CSF pressures, the timecourse of
improvement is inconsistent with a temporary CSF tap, and the one
child with a transient CSF leak showed less improvement. In
summary, intraventricular delivery of LPD complexes in these 2
children was safe and well tolerated. MRS, VEPs and
neurodevelopmental assessment suggest that gene transfer occurred.
Analysis of white matter signal in the repeated MRI is strongly
suggestive of remyelination in one child.
Example 6
Additional Human Clinical Trials
[0120] Nine additional children have been treated using the same
methodology (10 mls, same vector, administered intracerebral
ventrically). The data show safety and efficacy, as measured by
neurological improvements and normalization of evoked
potentials.
[0121] The following abbrieviations were used herein: NGF--nerve
growth factor; BDNF--brain derived neurotrophic factor; GDNF--glial
cell line-derived neurotrophic factor; GABA--gamma-amino butyric
acid; CNS--central nervous system; AAV--adeno-associated vector;
ITR--inverted terminal repeats; LPD--lipid-entrapped,
polycation-condensed DNA; bbb--blood-brain-barrier;
CMV--cytomegalovirus; ASPA--aspartoacyclase; GMP--guanosine
monophosphate; DOPE--dioleoyl phosphatidylethanolamine;
DC-Chol--3b-[N-(N',N' dimethylaminoethane)carbamoyl]cholesterol;
EGTA--ethylene glycol-bis(.beta.-aminoethyl ether);
ICC--immunocytochemical; PF--paraformaldehyde; GS--goat serum;
NHS--normal horse serum; PBS--phosphate buffered saline;
CSF--cerebral spinal fluid; NF--necrosis factor/nuclear
factor/nerve factor/neurofibromatosis; GFAP--glial fibrillary
acidic protein; NAA--N-acetylaspartate; H&E--hemotoxylin &
eosin; MHC--major histocompatibility compex; HSV--herpes simplex
virus; GPDS--Gesell-Provence Developmental Schedules; VEP--visual
evoked potentials; CHOP--Childrens' Hospital of Philadelphia;
NMRS--nuclear magnetic resonance scan; AEP--auditory evoked
potentials.
[0122] The teachings of the references cited herein are
specifically incorporated by reference.
* * * * *