U.S. patent application number 11/890761 was filed with the patent office on 2008-10-09 for compounds and methods to enhance raav transduction.
This patent application is currently assigned to University of Iowa Research Foundation. Invention is credited to John F. Engelhardt, Keith L. Munson, Ziying Yan.
Application Number | 20080249050 11/890761 |
Document ID | / |
Family ID | 33162204 |
Filed Date | 2008-10-09 |
United States Patent
Application |
20080249050 |
Kind Code |
A1 |
Engelhardt; John F. ; et
al. |
October 9, 2008 |
Compounds and methods to enhance rAAV transduction
Abstract
Agents and methods to alter rAAV transduction are provided.
Inventors: |
Engelhardt; John F.; (Iowa
City, IA) ; Munson; Keith L.; (Seattle, WA) ;
Yan; Ziying; (Iowa City, IA) |
Correspondence
Address: |
Schwegman, Lundberg, Woessner & Kluth, P.A.
P.O. Box 2938
Minneapolis
MN
55402
US
|
Assignee: |
University of Iowa Research
Foundation
Targeted Genetics Corporation
|
Family ID: |
33162204 |
Appl. No.: |
11/890761 |
Filed: |
August 7, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10815262 |
Mar 31, 2004 |
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11890761 |
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60459323 |
Mar 31, 2003 |
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60512347 |
Oct 16, 2003 |
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Current U.S.
Class: |
514/44R |
Current CPC
Class: |
A61P 7/04 20180101; C12N
15/86 20130101; A61P 31/04 20180101; G01N 33/6893 20130101; A61P
11/00 20180101; A61P 25/00 20180101; G01N 33/6872 20130101; A61K
48/00 20130101; C12N 2750/14143 20130101; G01N 33/502 20130101;
A61P 3/06 20180101; A61P 35/00 20180101; G01N 33/5008 20130101;
A61P 43/00 20180101; A61P 25/16 20180101; A61P 3/12 20180101 |
Class at
Publication: |
514/44 |
International
Class: |
A61K 31/711 20060101
A61K031/711; A61P 43/00 20060101 A61P043/00 |
Goverment Interests
STATEMENT OF GOVERNMENT RIGHTS
[0002] This invention was made, at least in part, with a grant from
the Government of the United States of America (grant HL58340 from
the National Institutes of Health). The Government may have certain
rights in the invention.
Claims
1-32. (canceled)
33. A method to inhibit or treat a condition associated with
aberrant expression of an endogenous gene product, comprising:
contacting a mammal at risk of or having the condition, with an
effective amount of at least one agent that enhances AAV
transduction and an effective amount at least one rAAV comprising a
transgene encoding at least a portion of a functional gene product,
the expression of which in the mammal inhibits or treats at least
one symptom of the condition, wherein the agent is a
chemotherapeutic, a lipid lowering agent, an antibiotic or a food
additive.
34. The method of claim 33 wherein the aberrant expression is the
lack of or reduced expression of the endogenous gene product.
35. The method of claim 33 wherein one rAAV comprises a first
recombinant DNA molecule comprising linked: i) a first DNA segment
comprising a 5' ITR of AAV; ii) a second DNA segment comprising a
heterologous DNA; and iii) a third DNA segment comprising a 3' ITR
of AAV.
36. The method of claim 35 further comprising a second rAAV
comprises a second recombinant DNA molecule comprising linked: i) a
first DNA segment comprising a 5' ITR of AAV, and ii) a second DNA
segment comprising a heterologous DNA which has sequences which are
different than the sequences in the second DNA segment of the first
recombinant DNA molecule; and iii) a third DNA segment comprising a
3' ITR of AAV.
37. The method of claim 36 wherein the second DNA segment of the
first recombinant DNA molecule comprises a portion of an open
reading frame for a gene product, optionally linked to a
transcriptional regulatory element, and a splice donor site 3' to
the portion of the open reading frame, wherein the second DNA
segment of the second recombinant DNA molecule comprises a splice
acceptor site 5' to the remainder of an open reading frame, which
together with the second DNA segment of the first recombinant DNA
molecule encodes a functional gene product.
38. The method of claim 37 wherein the transcriptional regulatory
element is a promoter.
39. The method of claim 36 wherein the second DNA segment of the
first recombinant DNA molecule comprises an enhancer and the second
DNA segment of the second recombinant DNA molecule comprises an
open reading frame encoding a functional gene product.
40. The method of claim 36 wherein the second DNA segment of the
first recombinant DNA molecule comprises a promoter and the second
DNA segment of the second recombinant DNA molecule comprises an
open reading frame encoding a functional gene product.
41. The method of claim 33 wherein the expression of the transgene
is enhanced.
42. The method of claim 33 wherein the transgene encodes cystic
fibrosis transmembrane conductance regulator, .beta.-globin,
.gamma.-globin, tyrosine hydroxylase, glucocerebrosidase, aryl
sulfatase A, factor VIII, dystrophin or erythropoietin.
43-50. (canceled)
51. The method of claim 33 wherein at least one agent modulates
microfilaments or microtubules.
52. The method of claim 33 wherein at least one agent modulates
rAAV endocytosis.
53. The method of claim 33 wherein at least one agent modulates
rAAV trafficking in the cell.
54. The method of claim 33 wherein at least one agent modulates
rAAV processing in the cell.
55. The method of claim 33 wherein at least one agent modulates
rAAV nucleic acid degradation in the cell.
56. The method of claim 33 wherein at least one agent modulates
rAAV protein degradation in the cell.
57. The method of claim 33 wherein at least one agent modulates
rAAV transport to the nucleus.
58. The method of claim 33 wherein at least one agent modulates
viral genome transport to the nucleus.
59. The method of claim 33 wherein at least one agent modulates
subcellular localization of proteosomes.
60. The method of claim 33 wherein at least one agent is
epoxomicin, doxorubicin, doxil, daunorubicin, idarubicin,
epirubicin, aclarubicin, simvastatin or tannic acid.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation under 37 CFR 1.53(b) of
U.S. application Ser. No. 10/815,262 filed Mar. 31, 2004, which
claims the benefit of the filing date of U.S. application Ser. No.
60/459,323, filed Mar. 31, 2003, and of U.S. application Ser. No.
60/512,347, filed Oct. 16, 2003, the disclosures of which are
incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0003] Recombinant adeno-associated virus (rAAV) has several
characteristics that underscore its potential as a gene therapy
vector for numerous target organs and inherited or acquired
diseases, a vaccine vector or for diagnostics. Moreover, rAAV
vector systems potentially offer major advantages over other gene
delivery vehicles, including adenoviruses and retroviruses. These
include the ability of rAAV to readily transduce non dividing or
slowly dividing cells and persist essentially for the lifetime of
the cell, the lack of undesirable cellular immune responses since
all viral genes can be deleted from the vector, and the fact that
AAV has never been associated with human disease.
[0004] A serotype 2 rAAV (rAAV-2) vector expressing the CFTR gene
was the first AAV vector to be utilized in clinical trials. This
vector has demonstrated promise in patients with cystic fibrosis
and has advanced to phase II trials (Flotte et al., 1996; Wagner et
al., 1999; Wagner et al., 2002; Aitken et al., 2001). In recent
years, additional rAAV 2 vectors have been or are currently being
advanced to clinical trials to treat a number of disease states
including a rAAV2 factor IX vector in phase I clinical trials in
patients with hemophilia B (Kay et al., 2000), and a rAAV2
sarcoglycan vector in phase I clinical trials of patients with CNS
disease. Additionally, clinical trials for rAAV expressing proteins
to treat Parkinsons disease (RDAC, 2001), and Canavan's disease
(Janson et al., 2001), have been proposed.
[0005] Other serotypes of AAV are known to exist, although they are
all closely related at the functional, structural, and genetic
level (see, e.g., Blacklow, 1988; and Rose, 1974). All AAV
serotypes exhibit very similar replication properties mediated by
homologous rep genes; all bear three related capsid proteins such
as those expressed in AAV-2, and all contain 5'-3' ITR sequences.
Currently, 8 serotypes of AAV have been described with the complete
genome sequence information available for AAV-1-AAV-6 (Srivastava
et al., 1983; Miramatsu et al., 1996; Chiorini et al., 1967; Xiao
et al., 1999; Chiorini et al., 1999; Bantel-Schaal et al., 1999;
Rutledge et al., 1998) and capsid gene sequence for AAV-7 and AAV-8
(Gao et al., 2002). AAV-6 has been shown to be a recombinant
between AAV-1 and AAV-2. In addition, there are two isolates and
sequences of AAV-3 that differ from each other in a number of amino
acids in both rep and cap (Rutledge et al., 1998). AAV-5 is the
most distantly related of the serotypes, and displays a
serotype-specific terminal resolution site (trs) in its ITR
(Chiorini et al., 1999). Even though rep proteins from other
serotypes bind the AAV-5 ITR, they do not efficiently cleave at the
trs. In addition to recent developments in AAV and rAAV serotypes,
numerous groups are experimenting with rAAV pseudotypes.
[0006] Variations in cell surface receptor usage for binding of
rAAV to cell membranes exists among various serotypes and may be in
part responsible for the differences in transduction efficiencies
in various tissue and cell types. Although conflicting data exists,
it has become apparent that there are differences among the
serotypes in the efficiency of transgene expression in various
tissues and cell types. For example, rAAV-1 and rAAV-7 overall
appear several orders of magnitude superior for transduction of
murine muscle tissue although rAAV-5 also demonstrates enhancement
compared to rAAV-2 (Gao et al., 2002; Chao et al., 2000; Rabinowitz
et al., 2002; Hildinger et al., 2001). rAAV-8 transduces murine
liver up to 100-fold higher (Gao et al., 2002) than rAAV-2, and
rAAV-5 appears superior in transduction of cells of the murine
respiratory tract (Zabner et al., 2000; Aurichio et al., 2002).
rAAV-5 generally appears to be superior to rAAV-2-based vectors in
all tissue types tested so far including CNS, muscle, liver and
retina (Chao et al., 2000; Rabinowitz et al., 2002; Hildinger et
al., 2001; Aurichio et al., 2002; Davidson et al., 2000; Mingozzi
et al., 2002). Similarly, rAAV-6 is more efficient than rAAV-2 in
transducing murine airway epithelia and alveoli, while rAAV-3 is
superior in transducing smooth muscle cells (Halbert et al., 2001;
Halbert et al., 2000). rAAV-4 transduces ependymal cells in the
murine CNS almost exclusively, while rAAV-5 transduces both neurons
and astrocytes (Davidson et al., 2000). In retina, a number of
studies have demonstrated large differences among serotypes in the
ability to transduce photoreceptor cells and the retinal pigmented
epithelium (Walters et al., 2001; Aurricchio et al., 2001; Yang et
al., 2002).
[0007] Despite the fact that rAAV has a very broad host tropism in
a variety of human, simian, and rodent cell lines (Lebkowski et
al., 1988; Muzyczka, 1992), the overall transduction efficiency in
human airway epithelia and other tissues seems to be quite low.
Previous studies have suggested that single to double strand
conversion of the rAAV genome may be the rate-limiting step for
AAV-mediated gene transfer (Ferrari et al., 1996; Fisher et al.,
1996). These studies demonstrated that adenovirus E4orf6 enhances
the conversion of single-stranded DNA genomes to linear,
double-stranded replication form dimers (Rfd) and monomers (Rfm),
through a pathway characteristic of the lytic phase of rAAV
replication. The structure of these replication forms consists of
head-to-head and tail-to-tail orientated linear concatamers with
one covalently linked end (Ferrari et al., 1996; Fisher et al.,
1996). In contrast, recent studies have elucidated an alternative
pathway for the conversion of rAAV genomes to double-stranded
circular intermediates with head-to-tail monomer and concatamer
structures (Duan et al., 1999; Duan et al., 1998; Sanlioglu et al.,
1999). The distinct pathways leading to the formation of either
circular AAV genomes or Rf intermediates appear to be regulated by
different cellular factors. For example, adenoviral E4orf6
expression decreases circular genome formation while adenovirus E2a
enhances its formation (Duan et al., 1999). Similarly, UV
irradiation also enhances AAV circular intermediate formation but
not Rf intermediates (Sanlioglu et al., 1999).
[0008] More recently, when cellular binding protein FK506-(FKBP-52)
was phosphorylated at tyrosine residues (by the epidermal growth
factor receptor protein tyrosine kinase), FKBP-52 was demonstrated
to be bound to the single-stranded D-sequence of the AAV ITR
causing an impairment in second strand synthesis (Qing et al.,
2001; Qing et al., 2003. The efficiency of rAAV transduction in a
number of cell types in vitro and in vivo correlates with the
phosphorylation state of FKBP-52. For example, in HeLa cells,
overexpression of a cellular phosphatase (TC-PTP), led to
dephosphorylation of the FKBP-52, an increase in AAV second-strand
DNA synthesis, and an increase in transgene expression. Transgenic
mice expressing either the wild type (wt) or a catalytically mutant
form of TC-PTP, were created. Hematopoietic stem cells from
transgenic mice expressing the wt TC-PTP phosphatase were
transduced by a rAAV2, but those from the phosphatase-negative
mutant were not. These results suggest that the block to
second-strand DNA synthesis is due to binding of FKBP-52 to the
D-sequence of infecting vector genomic DNA and that this binding is
regulated by phosphorylation. Thus, numerous strategies aimed at
increasing the transduction frequency for AAV have focused on
enhancing the molecular conversion of nonfunctional viral genomes
to expressible forms (Fisher et al., 1996; Sanlioglu et al., 1999)
or by increasing transcription and translation efficiencies by
altering the transgene expression cassettes (Zabner et al., 1996;
Xiao et al., 1998).
[0009] A second approach aimed at improving transduction
efficiencies of rAAV has focused on the binding of rAAV to cell
surface receptors. Many primary and secondary cell surface receptor
molecules have been identified for the various AAV serotypes. The
primary receptors identified (heparin sulfate and sialic acid) are
found on many cell types and are also utilized by a large number of
viruses besides AAV. This suggests that additional receptors that
lend more specificity to attachment and penetration of cells might
exist and several such co-receptors have been identified. Thus,
additional strategies to improve rAAV transduction efficiency have
focused on manipulation of cell surface receptors (Qing et al.,
1997) and/or receptor ligands in the virus coat proteins (Wickham
et al., 1996a; Wickham et al., 1996b; Bartlett et al., 1999).
[0010] While binding to the cell surface membrane and successful
conversion to a double stranded DNA genome are important, the
efficiency of these events does not necessarily correlate with the
overall ability or efficiency of rAAV to transduce a given cell
type. This has been increasingly apparent in recent years as a more
detailed understanding of the trafficking and uncoating of rAAV has
been accumulated (Duan et al., 1998; Seisenberger et al., 2001;
Hanson et al., 2001; Bantel-Schaal et al., 2002; Yan et al., 2002).
For example, polarized human airway epithelial cells are transduced
with varying efficiencies by rAAV-2 depending on the route of
delivery; entry from the basolateral surface results in about a
200-fold increase in gene expression in the cells compared to
vector administered from the apical surface (Duan et al., 1998;
Duan et al., 2000). Surprisingly, the difference in rAAV cell
surface binding between two cell surfaces is only about 5-fold.
This finding led to the discovery that the vectors traffic
differently in these cells depending on the route of delivery (Duan
et al., 1998; Duan et al., 2000).
[0011] Previous reports have clearly demonstrated that
intracellular trafficking to the nucleus for rAAV-2 and canine
parvovirus is a slow, rate-limiting process for certain cell types
(Parker et al., 2000; Hanson et al., 2001; Hanson et al., 2000;
Duan et al., 2000). Canine parvovirus and rAAV-2 have also been
demonstrated to be endocytosed through clathrin-dependent receptor
endocytosis and processed through endosomal compartments in a
similar fashion to transferrin, but not a fluid phase marker such
as dextran (Parker et al., 2000; Bartlett et al., 2000; Benson et
al., 2000; Duan et al., 1999). Transferrin trafficking has been
extensively studied and shown to move through the early endosome to
perinuclear recycling endosome (PNRE) (Sonnichsen et al., 2000; Ren
et al., 1998). The recycling of transferrin through the PNRE
requires the coordinated interactions of several small GTPases
(Rab5, Rab4, and Rab11) which direct the movement and fusion of
early endosomes to the PNRE compartment (Sonnichsen et al.,
2000).
[0012] Studies designed to develop agents to improve the efficiency
of rAAV transduction have demonstrated that proteosome inhibitors
such as the tripeptides LLnL and Z-LLL can enhance transduction of
rAAV. Agents of this class affect ubiquitination of rAAV by
inhibiting calpains, cathepsins, cysteine proteases as well as the
chymotrypsin-like protease activity of proteasomes in polarized
cell types (Duan et al., 2000; Yan et al., 2002). Additionally,
agents affecting DNA metabolism including hydroxyurea, novobiocin,
amsacrine, and etopside were tested for the ability to enhance rAAV
transduction based on the hypothesis that these drugs would
increase the rate of conversion of the single stranded rAAV genome
to a double stranded form. Results demonstrated that etoposide,
hydroxyurea, and campothecin were effective at enhancing rAAV
transduction when utilized singularly but when used in combination
produced no additive or synergistic effects (Russel et al., 1995).
Furthermore, these agents were only stated as effective in
enhancing rAAV transduction in cell types for which gene conversion
is rate limiting. Steps which proceed gene conversion (i.e.,
intracellular trafficking and processing of virions) appear to be
critical rate-limiting steps in primary cells and differentiated
tissues such as the airway (Hansen et al., 2000; Duan et al.,
2000).
[0013] There exists a need for improved transduction efficiencies
for rAAV vectors. Thus, what is needed is the identification of
agents that can alter, e.g., increase or enhance, rAAV transduction
or rAAV transduction frequencies in vivo. What is also needed is
the identification of agents that increase or enhance the
expression of a rAAV heterologous transgene in non-dividing or
slowly dividing cells or tissues, such as those in the liver and
the airway.
SUMMARY OF THE INVENTION
[0014] The invention provides a method to identify an agent, or a
combination of agents, that alters adeno-associated virus (AAV)
transduction of a eukaryotic cell, e.g., a mammalian cell such as a
mammalian lung or liver cell, or a population of eukaryotic cells,
e.g., in tissues or organs. For example, the invention provides a
method to identify agents that enhance rAAV transduction, e.g.,
enhance rAAV endocytosis, enhance trafficking and processing of the
rAAV through the intracellular compartments, including without
limitation proteosomes, endosomes, and trans-golgi, decrease viral
nucleic acid or protein degradation, increase viral uncoating
and/or increase nuclear transport of virus or the viral genome,
e.g., via cytoskeletal components such as microtubules or
microfilaments. The method comprises contacting the cell or
population of cells with one or more agents and virus. Then it is
determined whether virus transduction is altered. Preferred cells
include those of mammals, birds, fish, and reptiles, especially
domesticated mammals and birds such as humans, non-human primates,
cattle, sheep, pigs, horses, dogs, cats, mice, rats, rabbits,
chickens, and turkeys. For example, polarized human airway
epithelial cells grown at an air-liquid interface or human
bronchial xenografts are useful to identify agents which alter
viral transduction.
[0015] In one embodiment, agents to be tested are selected from
agents including those having desirable properties, e.g.,
therapeutic properties or functional and/or structural properties
of other agents identified as altering rAAV transduction. An agent
or library of agents may be randomly screened in the methods of the
invention. Alternatively, agents to be tested may be selected from
agents having desirable properties for a particular cell type,
tissue type or disease type to be treated with rAAV. Moreover,
agents may be selected from agents that modulate the proteosome,
e.g., agents that bind to a proteosome, alter the interaction of
virus and the proteosome, alter a function of the proteosome,
stabilize the proteosome, or alter the trafficking of the
proteosome, but do not inhibit the proteolytic activity of the
proteosome. As used herein, agents that are "proteosome modulating
agents" do not include agents that inhibit the proteolytic activity
of the proteosome. For example, to identify an agent useful to
enhance transduction of a CFTR rAAV vector for delivery to the
lungs of patients with cystic fibrosis, agents may be selected from
agents used or approved for use in cystic fibrosis patient
populations, agents in clinical trials or having FDA approval,
and/or agents associated with viral transduction, e.g., rAAV
endocytosis, trafficking and processing of the rAAV through
intracellular compartment(s), e.g., endosomal compartments,
decreased viral nucleic acid or protein degradation, increased
viral uncoating, or increased nuclear transport of virus or the
viral genome, agents that interact with cytoskeletal elements,
e.g., microtubules or microfilaments. In one embodiment, the agent
is not an agent inhibits proteolytic activity of the proteosome. In
one embodiment, the agent alters, e.g., enhances, transduction of a
mammalian cell by rAAV after viral binding to the cell membrane and
before second strand synthesis which yields an expressible form of
the viral genome. Randomized screening may be performed using a
transgene expressing rAAV, e.g., a reporter transgene encoding GFP,
or high throughput screening of chemical libraries on indicator
cell lines. Transduction may be assessed using expression of the
rAAV encoded reporter transgene. In one embodiment, chemical
libraries are selected based on chemical structures known to
interact with the proteasome, virus, or other intracellular
processing pathways, e.g., endosomal compartments, through which
virus is processed. Alternatively, peptide libraries may be
screened to identify agents that enhance rAAV transduction, for
instance, via an interaction with a proteosome that affects rAAV
transduction.
[0016] The agents of the inventions may be tested and/or used with
any serotype or pseudotype of rAAV vectors. It is envisioned that
agents identified as enhancing rAAV transduction may function with
all AAV serotypes and pseudotypes although there may be variations
in the degree of enhancement, cell or tissue type specificities or
concentrations employed for enhancement.
[0017] Agents of the invention may be used alone or in combination
to produce additive or synergistic transduction effects, to
increase the efficiency of transduction for multiple cell or tissue
types, to increase the time period of rAAV heterologous transgene
expression, to shorten the time period to expression of the
transgene, or to reduce the amount of virus needed to achieve a
therapeutic or prophylactic effect compared to transduction by or
expression of the same vector in the absence of the agent or
agents, or when an agent is used singularly. It is also
contemplated that one or more agents of invention may be used in
combination with agents that increase binding to cellular
receptors, promote conversion of the single stranded rAAV vector to
the double stranded form, or inhibit proteosome proteolytic
functions, to produce additive or synergistic transduction effects,
to increase the efficiency of transduction for multiple cell or
tissue types, to increase the time period of rAAV heterologous
transgene expression, to decrease lag time between contact of the
host cell with rAAV and expression of the transgene, or to reduce
the amount of virus needed to achieve a desired effect, compared to
transduction by or expression of the same vector in the absence of
the agent, a single agent or less than all of the agents. Agents of
the invention used in combination may be synergistic or additive in
enhancing rAAV transduction. Examples of additive effects of rAAV
transduction include, e.g., a shortened lag time between infection
and expression of the transgene and an overall longer time period
of expression.
[0018] Accordingly the invention provides a method to enhance rAAV
transduction of a mammalian cell. The method includes contacting
the mammalian cell with at least one rAAV and at least two agents,
e.g., in an amount effective to additively or synergistically
enhance rAAV transduction.
[0019] Agents of the invention may be employed with a rAAV vector
that contains only a single heterologous transgene, i.e., one not
derived from AAV sequences, e.g., not encoding a AAV protein, or
with dual vector strategies wherein the rAAV vectors contain more
than one heterologous transgene and/or transcriptional regulatory
elements as described in Duan et al. (2000); Yan et al. (2000); and
Duan et al. (2001). The gene being expressed can be either a DNA
segment encoding a polypeptide, catalytic RNA, or antisense RNA,
with whatever control elements (e.g., promoters, operators) are
desired.
[0020] The invention includes agents that modulate proteosomes
including agents that bind to, and/or alter one or more activities
of a proteosome, the association of virus with the proteasome,
and/or subcellular positioning of the proteasome. Proteosomes are
the main proteolytic complex in the cytosol and nucleus, and can be
transported between the cytoplasm and nucleus. For instance, the
26S proteosome complex comprises a 19S regulatory unit and a 20S
catalytic core which has chymotrypsin-like activity, i.e., cleavage
after large hydrophobic residues, trypsin-like activity, i.e.,
cleavage after basic residues, post-glutamyl hydrolase activity,
i.e., cleavage after acidic residues, branched amino acid cleavage
activity and small neutral amino acid cleavage activity. As
described herein, doxorubicin, an approved antibiotic, also
enhances rAAV transduction. Doxorubin may facilitate viral binding
to the proteasome and/or subsequent transportation into the
nucleus. In contrast, proteasome inhibitors such as LLnL and Z-LLL
more significantly inhibit core proteolytic activity of the
proteasome.
[0021] Hence, the combined use of agents that individually have
different or overlapping properties that alter rAAV transduction,
as well as agents with similar or identical properties, can result
in an additive and/or synergistic effect and so enhance rAAV
transduction. Thus, agents that enhance virus transduction are
particularly useful in gene therapy that employs rAAV to introduce
and/or express a therapeutic peptide or polypeptide, or in vaccines
that employ rAAV to introduce and/or express an immunogenic
prophylactic polypeptide or peptide, such as one from a virus,
fungus, bacterium, yeast or cancer cell, so as to induce an immune
response to that polypeptide or peptide. The agents of the
invention are also useful for the development of diagnostic markers
to aide in in vivo cellular marking of cells or tissue, or to track
and/or target chemotherapeutic strategies. Further, agents of the
invention may enhance rAAV transduction of cells, tissues, or
animals employed for the production of therapeutic proteins, e.g.,
growth hormone, cytokines or other recombinant proteins.
[0022] Further, the cells to be transduced may be contacted with
one or more agents prior to viral infection, concurrently with
viral infection, subsequent to viral infection, or any combination
thereof. Cells to be transduced may be contacted with one or more
agents at a single time point, e.g., a single dose of one agent or
a single dose of two or more agents, or at multiple time points, as
described above, e.g., multiple doses of one agent, multiple doses
of a combination of two or more agents, sequential or alternating
doses of two or more agents.
[0023] As described hereinbelow, virus binding, e.g., the
restricted distribution of viral receptors, and endocytosis of
AAV-2 at the apical membrane of airway epithelia is not the major
rate limiting step in transduction of this tissue type. In fact,
differentiated human airway epithelia internalize rAAV-2 quite
efficiently from the apical surface. Rather, endosomal processing
and trafficking of internalized virus to the nucleus is the major
obstacle encountered by AAV-2 following infection from the apical
membrane of the airway. In contrast to basolateral infection that
led to the efficient conversion of single stranded AAV DNA to
circular form genomes, apical infection gave rise to persistent
intracellular single stranded viral DNA in a transcriptionally
inactive state for up to 50 days. Using proteasome inhibitors which
increase the efficiency of endosomal processing of AAV-2 and
intracellular routing to the nucleus, a significantly enhanced
transduction from the apical surface of more than 200-fold was
observed, to nearly that of transduction from the basolateral
surface. It was also found that AAV capsid proteins are
ubiquitinated following endocytosis, and that ubiquitin-mediated
proteasome degradation of incoming virus can be blocked by
treatment with either proteasome or ubiquitin ligase
inhibitors.
[0024] Moreover, importantly, in vivo application of proteasome
inhibitor in mouse lung augmented rAAV gene transfer from
undetectable levels to a mean of 10.4+/-1.6% of the epithelial
cells in large bronchioles. Thus, the use of one or more agents
that alter rAAV endocytosis, trafficking and processing of the rAAV
through the intracellular compartments, including without
limitation proteosomes, endosomes, and trans-golgi, viral nucleic
acid or protein degradation, viral uncoating and/or nuclear
transport of virus or viral genome, e.g., via cytoskeletal
components such as microtubules or microfilaments, to circumvent
the major endosomal processing barriers to transduction in the
airway may provide clinically useful strategies for in vivo
AAV-mediated gene therapy of respiratory disorders such as cystic
fibrosis, as well as for other tissues in which viral processing
appears to be a rate limiting event, or strategies for in vivo
rAAV-mediated vaccines.
[0025] As also described hereinbelow, the transduction efficiency
of a recombinant AAV-2 construct with an RSV LTR promoter driving a
luciferase reporter that was packaged into both AAV-2 and AAV-5
capsid particles was compared in a number of cell lines and in lung
in vivo. Co-administration of the viruses with agents of the
invention including a proteosome modulator and/or a proteosome
inhibitor in vitro not only increased the transduction efficiency
of AAV-2, it also augmented AAV-5 mediated gene transfer although
often to a slightly lower extent. Increased transgene expression in
the presence of proteasome inhibitor was independent of viral
genome degradation since no significant difference of the amount of
internalized viral DNA was detected 24 hours after infection.
Western blot assays of immunoprecipitated viral proteins from
infected HeLa cell lysates and in vitro reconstitution experiments
revealed evidence for ubiquitin conjugation of both AAV-2 and AAV-5
capsids. These studies suggest that the previously reported barrier
involving the ubiquitin/proteasome pathway for rAAV-2 is also
active for rAAV-5 capsid entry pathways. In vivo co-administration
of a pseudotyped rAAV and the proteosome inhibitor Z-LLL induced
whole lung luciferase expression 17.2- and 2.1-fold at 14 and 42
days post-infection, respectively, while co-administration of rAAV
and a different rAAV transduction enhancing agent, doxorubicin,
induced tracheal and bronchial luciferase expression at higher
levels at 14, 42 and 90 days post-infection relative to expression
levels induced by Z-LLL. Surprisingly, at 42 days, luciferase
expression in trachea and bronchi in mice co-administered virus and
Z-LLL and doxorubicin was more than additive when administered by
endotracheal instillation. In human polarized airway epithelia,
combined doxorubicin and LLnL enhanced luciferase expression from
rAAV synergistically more that 1000-fold while individually
doxorubicin and LLnL enhanced transduction by 100- and 10-fold,
respectively. As also described herein, other agents that bind to
proteosomes and/or modulate proteosome activity, can alter rAAV
transduction efficiency.
[0026] Further, the activity of agents that alter virus
transduction, e.g., a modulator of rAAV endocytosis, trafficking
and processing of rAAV through the intracellular compartment, viral
nucleic acid or protein degradation, viral uncoating and nuclear
transport, may be enhanced by the addition of agents, such as EDTA
or EGTA, which may alter molecules in pathways associated with
virus transduction, e.g., agents such as calcium chelators or
modulators of intracellular calcium levels. Thus, one or more
agents that alter virus transduction may be employed with an agent
that enhances the activity of, or acts synergistically with, the
one or more agents that alter virus transduction. Thus, the
invention also provides for compositions or kits comprising two or
more agents, e.g., a first agent that alters virus transduction and
a second agent which enhances the activity of the first agent or
acts synergistically with the first agent.
[0027] Therefore, the invention also provides a method to alter
adeno-associated virus transduction of a eukaryotic cell or a
population of cells. The method comprises contacting the cell or a
population of cells with an amount of virus and an amount of at
least one agent of the invention effective to alter virus
transduction. The agent may be contacted with the cell concurrently
with the virus, prior to contacting the cell with virus or after
contacting the cell with virus. In one embodiment, the invention
provides a method in which a mammalian cell is contacted with at
least one rAAV and at least one agent that alters rAAV endocytosis,
rAAV trafficking or processing in intracellular compartments, viral
nucleic acid or protein degradation, and/or nuclear transport of
virus or viral genomes. In one embodiment, the agent is not an
inhibitor of proteosome proteolytic activity. The agent(s) and/or
virus may each be administered once, or in repeated dosing, so as
to achieve the desired effect, i.e., to enhance rAAV transduction.
Since AAV has been shown to have a broad host range (e.g., for
pulmonary expression) and persists in muscle, rAAV may be employed
to express a gene in any animal, and particularly in mammals,
birds, fish, and reptiles, especially domesticated mammals and
birds such as cattle, sheep, pigs, horses, dogs, cats, chickens,
and turkeys. Both human and veterinary uses are particularly
preferred.
[0028] In one embodiment, at least one rAAV and/or pseudotyped rAAV
and one or more agents of the invention may be employed in methods
to alter, e.g., increase, transduction efficiency and transgene
expression, methods to detect or determine transgene expression
efficiency, methods to screen for promoter strength and/or RNA
stability, as well as in therapeutic or prophylactic therapies in
which the vectors are useful include blood disorders (e.g., sickle
cell anemia, thalassemias, hemophilias, and Fanconi anemias),
neurological disorders, such as Alzheimer's disease and Parkinson's
disease, and muscle disorders involving skeletal, cardiac or smooth
muscle, as well as diseases of the lung, e.g., cystic fibrosis and
asthma. In particular, therapeutic genes useful in the vectors of
the invention include a .alpha.-globin gene, .beta.-globin gene,
.gamma.-globin gene, Factor VIII gene, Factor IX gene, cystic
fibrosis transmembrane conductance regulator gene (CFTR), Fanconi
anemia complementation group, dystrophin gene, an antisense gene,
low density lipoprotein (LDL) gene, tyrosine hydroxylase gene
(Parkinson's disease), glucocerebrosidase gene (Gaucher's disease),
arylsulfatase A gene (metachromatic leukodystrophies),
erythropoietin gene, as well as genes encoding immunogenic
polypeptides or peptide, such as those useful for vaccines, or
genes encoding other gene products such as other peptides,
polypeptides or proteins. In one embodiment, the rAAV encodes a
catalytic RNA, e.g., a ribozyme or siRNA, e.g., one useful to
decrease expression of a particular RNA expressed in a cell.
[0029] Also within the scope of the invention is the inclusion of
more than one open reading frame in a rAAV vector, i.e., a
plurality of genes may be present in an individual vector. Further,
as rAAV may form concatamers after infection, each monomer of that
concatamer may comprise a different gene, or a portion thereof.
Circularized intermediates of recombinant adeno-associated virus
may impart episomal persistence to linked sequences.
[0030] Further, co-infection with two or more different rAAV may,
through intermolecular recombination, yield a concatamer having one
or more copies of any particular rAAV. The implications of
intermolecular recombination of rAAV genomes to form a single
molecule, e.g., a nuclear episome, which may be a concatamer
comprising at least two different rAAV genomes, is particularly
relevant for gene therapy with rAAV, as large regulatory elements
and genes beyond the packaging capacity of rAAV can be brought
together by co-infecting cells or tissue of an organism with two
independent rAAV vectors. For example, enhancers and/or promoters
may be introduced into one vector while DNA comprising an open
reading frame, e.g., a gene of interest, with or without a minimal
promoter, is introduced into a second vector. Thus, after
co-infection with the two vectors, the transgene cassette size is
increased beyond that for a single AAV vector alone and the DNA
comprising the opening reading frame is linked to the enhancer
and/or promoter. In another embodiment of the invention, vectors
encoding two independent regions of a gene are brought together to
form an intact splicing unit. Without being bound by theory, agents
of the invention may increase concatamerization and/or
intermolecular recombination of rAAV by increasing the steady state
abundance of viral genomes resulting in enhanced transduction
frequencies of rAAV compared to cells not treated with agents of
the invention. Agents of the invention that alter processing of
rAAV virions in the cytoplasm and/or nucleus may also influence the
presentation of viral DNA in the nucleus and hence alter gene
conversion products. Such altered presentation may affect
concatamerization by allowing for more localized accumulation of
virions at specific sites within the nucleus. Alternatively,
ubiquitination of associated factors with viral DNA (i.e., Rep or
host cell proteins) may affect the biologic properties of these
associated factors and influence linear, circular, and/or
concatamerization processes. Thus, agents of this invention may
influence intramolecular concatamerization and the efficiency of
multiple vector technologies by the mechanisms discussed above.
[0031] Accordingly, the use of multiple rAAV vectors is useful to
overcome the current size limitation for transgenes within rAAV
vectors, and allows for the incorporation of a larger
transcriptional regulatory region, e.g., a stronger heterologous
promoter or an endogenous promoter, e.g., the CFTR endogenous
promoter, or one or more enhancer sequences.
[0032] Therefore, two or more, e.g., a plurality, of DNA segments,
each in an individual rAAV vector, may be delivered to a cell, so
as to result in a single DNA molecule having DNA segments from more
than one rAAV. In one embodiment of the invention, one rAAV may
comprise a first recombinant DNA molecule comprising linked: a
first DNA segment comprising a 5' ITR of AAV; a second DNA segment
which does not comprise AAV sequences (nonAAV sequences), i.e.,
heterologous sequences; and a third DNA segment comprising a 3' ITR
of AAV. A second recombinant AAV comprises a second recombinant DNA
molecule comprising linked: a first DNA segment comprising a 5' ITR
of AAV; a second DNA segment which does not comprise AAV sequences
and which second DNA segment has sequences which are different than
the sequences in the second DNA segment of the first recombinant
DNA molecule; and a third DNA segment comprising a 3' ITR of AAV.
One of the rAAV may be a pseudotype.
[0033] In one embodiment of the invention, one rAAV vector
comprises a first DNA segment comprising a 5' ITR linked to a
second DNA segment comprising the 5' end of an open reading frame
(but optionally not an entire open reading frame), optionally
operably linked to a promoter, e.g., a heterologous promoter, and a
5' splice site linked to a third DNA segment comprising a 3' ITR.
The second rAAV vector comprises a first DNA segment comprising a
5' ITR linked to a second DNA segment comprising a 3' splice site
and the 3' end (the remainder) of an open reading frame, i.e., the
second DNA segment of the second vector together with the second
DNA segment of the first vector encodes a functional gene product
linked to a third DNA segment comprising a 3' ITR. A "functional"
gene product is one which has a detectable activity or is capable
of having a detectable activity when present in an appropriate
cell, tissue or organism, e.g., has at least one activity, and
preferably substantially the same activity, as a reference, e.g.,
corresponding, gene product, for example, a wild-type (full-length)
polypeptide or ribozyme. Preferably, the second DNA segments
together comprise DNA encoding, for example, CFTR, factor VIII,
dystrophin, or erythropoietin. The second DNA segments may be
obtained or derived from cDNA, genomic DNA or a combination
thereof. For example, the second DNA segment of the first vector
may comprise one or more, but not all of the exons of a gene
comprising more than one exon and the second DNA segment of the
second vector may comprise at least one exon of the gene that is
not present in the first vector, or one or more exons from a
different gene (thereby coding for a chimeric polypeptide). The
second DNA segment of the first vector may comprise the endogenous
promoter of the respective gene, e.g., the epo promoter, or a
heterologous promoter.
[0034] In another embodiment, one rAAV vector comprises a first DNA
segment comprising a 5' ITR linked to a second DNA segment
comprising a promoter and/or enhancer linked to a third DNA segment
comprising a 3' ITR. Optionally, the first rAAV vector does not
include a splice donor and/or a splice acceptor. A second rAAV
vector comprises a first DNA segment comprising a 5' ITR linked to
a second DNA segment comprising at least a portion of an open
reading frame optionally linked to a promoter (a different promoter
than in the first vector or a second copy of the promoter in the
first vector) linked to a third DNA segment comprising a 3' ITR.
For example, the second DNA segment of the first recombinant DNA
molecule comprises at least one heterologous enhancer and/or at
least one heterologous promoter, i.e., the enhancer and/or promoter
sequences are not derived from AAV sequences. Preferably, the
second DNA segment of the second recombinant DNA molecule comprises
a portion of an open reading frame which encodes a functional gene
product.
[0035] In one embodiment, co-infection of a cell with at least one
pseudotyped rAAV, e.g., a transgene containing vector, and a second
vector comprising at least one, preferably at least two or more,
enhancer sequences, may result in an enhancement of transgene
expression from a minimal promoter. Furthermore, an enhancement can
also be achieved by cis-activation of ITRs in transgene-containing
vectors without a promoter. Thus, large regulatory elements
including tissue-specific enhancers can be introduced into cells by
a separate rAAV vector to regulate the expression of a second
transgene-containing AAV vector in cis following intracellular
concatamerization.
[0036] In yet another embodiment of the invention, the second DNA
segment of the first recombinant DNA molecule comprises a
cis-acting integration sequence(s) for a recombinase and also
encodes a recombinase or integrase that is specific for the
integration sequence(s), e.g., Cre/lox system of bacteriophage P1
(U.S. Pat. No. 5,658,772), the FLP/FRT system of yeast, the Gin
recombinase of phage Mu, the Pin recombinase of E. coli, the R/RS
system of the pSR1 plasmid, a retrotransposase or the integrase
from a lentivirus or retrovirus. The second DNA segment in the
second recombinant DNA molecule comprises at least a portion of an
open reading frame, and preferably a promoter operably linked to
the open reading frame. The formation of a concatamer comprising
the first and the second recombinant DNA molecules, and the
expression of the recombinase or integrase, will enhance the
integration of the concatamer, or a portion thereof, into the host
genome. Also, rAAV vectors comprising cis-acting integration
sequences and the corresponding recombinase or integrase are useful
to drive directional recombination, which, as discussed above, may
be particularly useful when employing two or more rAAV vectors.
[0037] Accordingly, the vectors of the invention are useful in a
method of delivering and/or expressing one or more genes in a host
cell, to prepare host cells having the vector(s), and in the
preparation of a composition comprising rAAV(s). A host cell may be
contacted with each rAAV individually, e.g., sequentially, with or
without an agent of the invention. To deliver the gene(s) to the
host cell, a recombinant adenovirus helper virus may be
employed.
[0038] Thus, the invention also provides a method to express a gene
product in a host cell. The host cell is preferably a mammalian
host cell, e.g., a murine, canine, feral or human cell, and may be
a lung, neuron or muscle cell. The method comprises contacting the
host cell with at least one rAAV vector and at least one agent of
the invention. In one embodiment, the host cell is contacted with
at least two different rAAV vectors. In one embodiment, one of the
rAAV vectors is a pseudotyped rAAV. The host cell is preferably
contacted with the vectors concurrently, although it is envisioned
that the host cell may be contacted with each vector at a different
time relative to the contact with the other vector(s), as discussed
herein. Two or more agents of the invention may also be employed in
the method, and may be contacted with the cell prior to, concurrent
with, or subsequent to contact of the cell with the vector(s). In
one embodiment, the agent modulates microfilament or microtubule
synthesis, formation or degradation, modulates rAAV endocytosis,
modulates rAAV trafficking in a cell, modulates rAAV processing in
a cell, modulates rAAV nucleic acid degradation in a cell,
modulates rAAV protein degradation in a cell, modulates rAAV
transport to the nucleus and/or modulates viral genome transport to
the nucleus. In one embodiment, two agents that modulate
microfilament or microtubule synthesis, formation or degradation,
rAAV endocytosis, rAAV trafficking in a cell, rAAV processing in a
cell, rAAV nucleic acid degradation in a cell, rAAV protein
degradation in a cell, rAAV transport to the nucleus and/or viral
genome transport to the nucleus, are employed.
[0039] Also provided is a method to detect expression of a
transgene in a cell. The method comprises contacting a host cell
with a rAAV of the invention which comprises a transgene comprising
a non-AAV promoter linked to an open reading frame, e.g., a marker
gene or an open reading frame having one or more genetic
modifications relative to a corresponding wild-type open reading
frame. One or more agents of the invention may also be employed in
the method, and may be contacted with the cell prior to, concurrent
with, or subsequent to contact of the cell with the vector(s). The
expression of the transgene is then detected or determined, e.g.,
relative to a host cell contacted with a rAAV comprising a
transgene linked to a different promoter or a transgene with the
same promoter but linked to a wild-type open reading frame or one
not contacted with an agent of the invention.
[0040] Thus, one embodiment, the invention provides a method to
prevent, inhibit or treat a condition associated with aberrant
expression of an endogenous gene product. The method includes
contacting a mammal at risk of or having the condition, with an
effective amount of at least one agent that enhances AAV
transduction and an effective amount at least one rAAV comprising a
transgene encoding at least a portion of a functional gene product,
the expression of which in the mammal inhibits the aberrant
expression of the corresponding endogenous gene product, e.g., via
a dominant negative, antisense or catalytic RNA, or encodes a
functional gene product, thereby preventing or inhibiting one or
more symptoms of the condition. In one embodiment, the agent is a
chemotherapeutic, a lipid lowering agent, an antibiotic or a food
additive. In one embodiment, the agent is not camptothecin. In
another embodiment, the agent is not cisplatin.
BRIEF DESCRIPTION OF THE FIGURES
[0041] FIGS. 1A-E. Luciferase activity in HeLa cells transfected
with rAAV FLAG-Luc in the presence or absence of various agents.
HeLa cells were contacted with 100 ppc AAV FLAG-Luc for 2 hours,
and cells were harvested 48 hours later. N=3, average .+-.standard
deviation.
[0042] FIG. 2. In vivo enhancement of rAAV transduction with Doxil.
Male Balb/c mice intravenously administered Doxil were
endotracheally instilled with 1.times.10.sup.11 DRP AAV2FLAG-Luc
(01:004)
[0043] FIG. 3. In vivo enhancement of rAAV transduction of Factor
VIII in Rag-1 mice treated with Doxil. Rag-1 mice intravenously
administered Doxil (20 mg/kg) were infected with 1.times.10.sup.12
DRP AAV5-FVIII, and Factor VIII levels at sacrifice determined.
Data are the average .+-.standard deviation.
[0044] FIG. 4. Luciferase activity in HeLa cells after infection
with A) AV2.RSVLuc or AV2.RSVlucCap5 (100 or 1000 ppc) or B)
AV2CMVluc or AV2CMVluc Cap5 (500 ppc), and co-administration of
LLnL (40, 200 or 400 .mu.M), Z-LLL (4 .mu.M), or doxorubicin (0.5,
1.0 or 5.0 .mu.M) or a combination of LLnL (4, 10, 20 or 200 .mu.M)
and doxorubicin (0.5, 1.0 or 5.0 .mu.M). C) Comparison of CMV and
RSV promoters in AAV-2 vectors in HeLa cells.
[0045] FIG. 5. Luciferase activity in A549 cells after infection
with AV2CMVluc or AV2CMVluc Cap5 (500 ppc), and co-administration
of LLnL (40, 200 or 400 .mu.M), Z-LLL (4 .mu.M), or doxorubicin (5
.mu.M). A) Comparison of AV2CMVluc and AV2CMVlucCap5. B) Dose
response for varying amounts of LLnL in A549 cells infected with
AV2CMVluc.
[0046] FIG. 6. Luciferase activity in ferret fibroblasts after
infection with AV2CMVluc or AV2CMVluc Cap5 (500 ppc), and
co-administration of LLnL (40, 200 or 400 .mu.M), Z-LLL (4 .mu.M),
or doxorubicin (1 .mu.M). A) Comparison of AV2CMVluc and
AV2CMVlucCap5. B) RLU at 1 and 5 days for AV2CMVluc and
AV2CMVlucCap5 in ferret fibroblasts.
[0047] FIG. 7. Comparison of luciferase activity in HeLa (A),
ferret fibroblast (B) and A549 (C) cells with one or two proteosome
modulators.
[0048] FIG. 8. Luciferase activity in polarized airway epithelial
cells at 3 days and 15 days after apical infection with
5.times.10.sup.9 AV2RSVLuc or AV2RSVlucCap5 and co-administration
of LLnL (40 .mu.M) or doxorubicin (1.0 or 5.0 .mu.M) or a
combination of LLnL (40 .mu.M) and doxorubicin (1.0 or 5.0
.mu.M).
[0049] FIG. 9. Luciferase activity in C57B16 mouse lung or trachea
and bronchi at 2 weeks (A) or 6 weeks (B) after infection (via
nasal aspiration) with AV2RSVlucCap5 (3 times with 10 .mu.l of
2.times.10.sup.9 particles/.mu.l in 40 .mu.l, for a total of
6.times.10.sup.10 particles) and co-administration of Z-LLL (200
.mu.M), doxorubicin (200 .mu.M), or a combination of Z-LLL (200
.mu.M) and doxorubicin (200 .mu.M). For each group, n=12. Lung and
trachea with some bronchial tissue was isolated and, after
extraction, luciferase activity/total protein in the tissue
extraction determined.
[0050] FIG. 10. Luciferase activity in mouse lung or trachea and
bronchi at 2 weeks, 6 weeks or 3 months after infection with
AV2RSVlucCap5 and co-administration of Z-LLL (200 .mu.M),
doxorubicin (200 .mu.M) or a combination of Z-LLL (200 .mu.M) and
doxorubicin (200 .mu.M). The luciferase assay was performed at 80%
sensitivity. Lung and trachea with some bronchial tissue was
isolated and, after extraction, luciferase activity/total protein
in the tissue extraction determined.
[0051] FIG. 11. The effects of proteasome inhibitors LLnL and
Doxorubicin (Dox) on AV2Luc and AV2/5Luc transduction of
immortalized human airway cell lines IB3 (panel A) and A549 (panel
B) were evaluated. Proteasome-modulating agents were
co-administered with each rAAV vector (MOI of 500 particles per
cell) at the time of infection and transduction was evaluated 24
hours later. Various concentrations of each chemical were evaluated
as indicated in each graph. Data represent the mean (+/-SEM)
relative luciferase activity experiment (N=4).
[0052] FIG. 12. LLnL and Dox both facilitate translocation of rAAV
to the nucleus. IB3 cells were infected with AV2eGFP (MOI=1000
particles/cell) in the presence or absence 40 .mu.M LLnL or 1 .mu.M
Dox. At 24 hours post-infection, cytoplasmic (Cyt) and nuclear
(Nuc) fractions were isolated. (A) Viral DNA in each fraction was
detected by slot-blot hybridization against a P.sup.32 labeled eGFP
probe and visualized using a BioRad phosphoimager (N=3 isolations
are shown for each condition). P.sup.32 signal was quantified using
BioRad software. (B) The percentage distribution of the signals in
nuclear and cytoplasmic fractions was calculated based on the mean
signal for the three experimental points. (C) Results of
S.sup.35-capsid labeled rAAV2 localization in polarized human
airway epithelia by in situ autoradiography. Infections were
performed in the presence and absence of LLnL treatment.
[0053] FIG. 13. Dox and LLnL provide additive induction of rAV2
transduction. Hela cells (left panel) and A549 cells (right panel)
were infected with rAAV (MOI 500 particles/cell) in the presence of
the indicated drug combinations and the expressed transgene was
assessed at 24 hours post-infection (Mean +/-SEM, N=4). Fold
induction relative to vehicle-treated rAAV-infected cells is
indicated above each bar.
[0054] FIG. 14. The effect of proteasome inhibitor LLnL on (A)
AV2Luc and (B) AV2/5Luc transduction was evaluated following apical
and basolateral infection of human polarized airway epithelia at an
MOI of 10,000 particles/cell in the presence and absence of LLnL
(40 .mu.M). Luciferase activity was measured at 5 and 14 days
post-infection. Values represent the mean (+/-SEM) relative
luciferase activity for three independent tissue samples (N=6-9
total transwells).
[0055] FIG. 15. Analysis of full-length and self-complementary
eGFP-expressing AAV vectors. HeLa cells were infected at an
MOI=1000 particles/cell. (A) Quantification of relative
eGFP-expressing area for AV2eGFP and scAV2eGFP vectors. The values
represent the mean (+/-SEM) for three independent infections and
quantification of 10 random fields for each experimental point. (B)
Response of AV2eGFP and scAV2eGFP vectors to treatment with
hydroxyurea (5 mM) with gene expression analyzed at 24 hours
post-infection. (C) Southern blot analysis of Hirt DNA harvested
from AV2eGFP-infected (lanes 1 and 2) and scAV2eGFP-infected (lanes
3 and 4) Hela cells at 24 hours post-infection. A .sup.32P-labeled
eGFP DNA probe was used for Southern blots.
[0056] FIG. 16. Quantification of eGFP expression following apical
infection of polarized human airway epithelia with
self-complementary and full-length eGFP vectors. The relative mean
area of fluorescence was evaluated following transduction with
AV2eGFP and scAV2eGFP vectors in the presence or absence of LLnL
(40 .mu.M) at an MOI of 10,000 particles/cell on 1, 3, 7, 15 and 30
days post-infection. The values represent the mean (+/-SEM) for
three independent tissue samples. For each tissue sample, 3
transwells were evaluated by imaging 10 random fields in each
sample at the various time points (N=9 total transwells for each
experimental point).
[0057] FIG. 17. Combined administration of proteasome-modulating
agents can synergistically induce rAAV transduction from the apical
surface of polarized human airway epithelia. (A) 1.times.10.sup.9
particles of AV2Luc were applied to the apical surface of polarized
human airway epithelia cultures in the absence and presence of
various combinations of LLnL (40 .mu.M) and/or Dox (5 .mu.M).
Luciferase expression was assayed at 3 and 17 days post-infection
(B-E). Similar results were observed following apical infection
with a self complementary (2.3 kb) scAV2eGFP vector at 15 days
post-infection. (F) Combined administration of LLnL and Dox
augments dual vector heterodimer-mediated delivery of a
trans-spliced LacZ gene product. 10.sup.10 particles of
AV2LacZdonor (indicated by D) and/or AV2LacZacceptor (indicated by
A) were used to infect each transwell of the polarized airway
epithelia in the presence or absence of co-administered LLnL (40
.mu.M) and Dox (5 .mu.M). .beta.-galactosidase activity was
evaluated at 15 days post-infection. Data represents the mean
(+/-SEM) relative luciferase or .beta.-galactosidase activity (per
1/10 sample) for 3 independent experiments.
[0058] FIG. 18. In vivo gene transfer to the mouse lung. AV2 and
AV2/5 luciferase vectors were used to evaluate the ability of
proteasome-modulating agents to induce transduction. Results depict
the mean (+/-SEM) luciferase expression from (N=5) mouse lungs at
14 days post-infection for each condition.
[0059] FIG. 19. Complementation of CFTR chloride transport
abnormalities in CF airway epithelia using combined CFTR rAAV and
proteasome inhibitor treatment. Results depict the mean +/-SEM
(N=9) delta Isc response to IBMX/forskolin in CF airway epithelia
treated under the indicated conditions. Assays were performed at 15
days post-infection and a non-CF untreated control is given as a
reference for fully functional CFTR.
[0060] FIG. 20. Screening for anthracycline proteosome modulators.
A) Graph of luciferase activity versus concentration of tested
agent. B) Fold change in luciferase activity for various
treatments.
[0061] FIG. 21. In vivo results for anthracycline proteosome
modulators.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0062] A "vector" as used herein refers to a macromolecule or
association of macromolecules that comprises or associates with a
polynucleotide and which can be used to mediate delivery of the
polynucleotide to a cell, either in vitro or in vivo. Illustrative
vectors include, for example, plasmids, viral vectors, liposomes
and other gene delivery vehicles. The polynucleotide to be
delivered, sometimes referred to as a "target polynucleotide" or
"transgene," may comprise a coding sequence of interest in gene
therapy (such as a gene encoding a protein of therapeutic or
interest), a coding sequence of interest in vaccine development
(such as a polynucleotide expressing a protein, polypeptide or
peptide suitable for eliciting an immune response in a mammal),
and/or a selectable or detectable marker.
[0063] "AAV" is adeno-associated virus, and may be used to refer to
the naturally occurring wild-type virus itself or derivatives
thereof. The term covers all subtypes, serotypes and pseudotypes,
and both naturally occurring and recombinant forms, except where
required otherwise. As used herein, the term "serotype" refers to
an AAV which is identified by and distinguished from other AAVs
based on capsid protein reactivity with defined antisera, e.g.,
there are eight serotypes of primate AAVs, AAV-1 to AAV-8. For
example, serotype AAV2 is used to refer to an AAV which contains
capsid proteins encoded from the cap gene of AAV 2 and a genome
containing 5' and 3' ITR sequences from the same AAV2 serotype.
Pseudotyped AAV as refers to an AAV that contains capsid proteins
from one serotype and a viral genome including 5'-3' ITRs of a
second serotype. Pseudotyped rAAV would be expected to have cell
surface binding properties of the capsid serotype and genetic
properties consistent with the ITR serotype. Pseudotyped rAAV are
produced using standard techniques described in the art. As used
herein, for example, rAAV5 may be used to refer an AAV having both
capsid proteins and 5'-3' ITRs from the same serotype or it may
refer to an AAV having capsid proteins from serotype 5 and 5'-3'
ITRs from a different AAV serotype, e.g., AAV serotype 2. For each
example illustrated herein the description of the vector design and
production describes the serotype of the capsid and 5'-3' ITR
sequences. The abbreviation "rAAV" refers to recombinant
adeno-associated virus, also referred to as a recombinant AAV
vector (or "rAAV vector").
[0064] "Transduction" or "transducing" as used herein, are terms
referring to a process for the introduction of an exogenous
polynucleotide, e.g., a transgene in rAAV vector, into a host cell
leading to expression of the polynucleotide, e.g., the transgene in
the cell. The process includes 1) endocytosis of the AAV after it
has bound to a cell surface receptor, 2) escape from endosomes or
other intracellular compartments in the cytosol of a cell, 3)
trafficking of the viral particle or viral genome to the nucleus,
4) uncoating of the virus particles, and generation of expressible
double stranded AAV genome forms, including circular intermediates.
The rAAV expressible double stranded form may persist as a nuclear
episome or optionally may integrate into the host genome. The
alteration of any or a combination of endocytosis of the AAV after
it has bound to a cell surface receptor, escape from endosomes or
other intracellular compartments to the cytosol of a cell,
trafficking of the viral particle or viral genome to the nucleus,
or uncoating of the virus particles, and generation of expressive
double stranded AAV genome forms, including circular intermediates,
by an agent of the invention, may result in altered expression
levels or persistence of expression, or altered trafficking to the
nucleus, or altered types or relative numbers of host cells or a
population of cells expressing the introduced polynucleotide.
Altered expression or persistence of a polynucleotide introduced
via rAAV can be determined by methods well known to the art
including, but not limited to, protein expression, e.g., by ELISA,
flow cytometry and Western blot, measurement of and DNA and RNA
production by hybridization assays, e.g., Northern blots, Southern
blots and gel shift mobility assays. The agents of the invention
preferably alter, enhance or increase viral endocytosis, escape
from endosomes or other intracellular cytosolic compartments, and
trafficking into or to the nucleus, uncoating of the viral
particles in the nucleus, and/or increasing concatamerization or
generation of double stranded expressible forms of the rAAV genome
in the nucleus, so as to alter expression of the introduced
polynucleotide, e.g., a transgene in a rAAV vector, in vitro or in
vivo. Methods used for the introduction of the exogenous
polynucleotide include well-known techniques such as transfection,
lipofection, viral infection, transformation, and electroporation,
as well as non-viral gene delivery techniques. The introduced
polynucleotide may be stably or transiently maintained in the host
cell.
[0065] "Increased transduction or transduction frequency", "altered
transduction or transduction frequency", or "enhanced transduction
or transduction frequency" refers to an increase in one or more of
the activities described above in a treated cell relative to an
untreated cell. Agents of the invention which increase transduction
efficiency may be determined by measuring the effect on one or more
transduction activities, which may include measuring the expression
of the transgene, measuring the function of the transgene, or
determining the number of rAAV vector particles necessary to yield
the same transgene effect compared to host cells not treated with
the agents.
[0066] "Proteosome modulator" refers to an agent or class of agents
which alter or enhance rAAV transduction or rAAV transduction
frequencies by interacting with, binding to, or altering the
function of, and/or trafficking or location of the proteosome.
Proteosome modulators may have other cellular functions as
described in the art, e.g., such as doxyrubicin, an antibiotic.
Proteosome modulators of the current invention do not include
proteosome inhibitors, e.g., such as tripeptidyl aldehydes (Z-LLL
or LLnL), agents that inhibit calpains, cathepsins, cysteine
proteases, and/or chymotrypsin-like protease activity of
proteasomes (Wagner et al., 2002; Young et al., 2000; Seisenberger
et al., 2001).
[0067] "Generation of double stranded expressible forms" or
"conversion of single to double strand rAAV genomes" refers to the
process of replicating in the nucleus of an rAAV infected host cell
a complimentary strand of the rAAV single stranded vector DNA
genome and annealing of the complimentary strand to the vector
genome to produce a double stranded DNA rAAV genome. Agents of the
invention described herein to increase, alter, or enhance rAAV
transduction include agents which increase the rate of nuclear
transport or the steady state of single stranded viral DNA genomes
in the nucleus which can drive gene conversion events via steady
state mechanisms. For the purposes of the invention described
herein, agents which enhance conversion of single to double strands
do not include agents which increase the concentration of DNA
repair enzymes or activate alternate DNA repair mechanism described
by Russel et al. (1995).
[0068] "Gene delivery" refers to the introduction of an exogenous
polynucleotide into a cell for gene transfer, and may encompass
targeting, binding, uptake, transport, localization, replicon
integration and expression.
[0069] "Gene transfer" refers to the introduction of an exogenous
polynucleotide into a cell which may encompass targeting, binding,
uptake, transport, localization and replicon integration, but is
distinct from and does not imply subsequent expression of the
gene.
[0070] "Gene expression" or "expression" refers to the process of
gene transcription, translation, and post-translational
modification.
[0071] A "detectable marker gene" is a gene that allows cells
carrying the gene to be specifically detected (e.g., distinguished
from cells which do not carry the marker gene). A large variety of
such marker genes are known in the art.
[0072] A "selectable marker gene" is a gene that allows cells
carrying the gene to be specifically selected for or against, in
the presence of a corresponding selective agent. By way of
illustration, an antibiotic resistance gene can be used as a
positive selectable marker gene that allows a host cell to be
positively selected for in the presence of the corresponding
antibiotic. A variety of positive and negative selectable markers
are known in the art, some of which are described below.
[0073] An "rAAV vector" as used herein refers to an AAV vector
comprising a polynucleotide sequence not of AAV origin (i.e., a
polynucleotide heterologous to AAV), typically a sequence of
interest for the genetic transformation of a cell. In preferred
vector constructs of this invention, the heterologous
polynucleotide is flanked by at least one, preferably two AAV
inverted terminal repeat sequences (ITRs). The term rAAV vector
encompasses both rAAV vector particles and rAAV vector
plasmids.
[0074] An "AAV virus" or "AAV viral particle" refers to a viral
particle composed of at least one AAV capsid protein (preferably by
all of the capsid proteins of a wild-type AAV) and an encapsidated
polynucleotide. If the particle comprises a heterologous
polynucleotide (i.e., a polynucleotide other than a wild-type AAV
genome such as a transgene to be delivered to a mammalian cell), it
is typically referred to as "rAAV".
[0075] A "rAAV vaccine" as used herein refers to an AAV vector
comprising a polynucleotide sequence not of AAV origin (i.e., a
polynucleotide heterologous to AAV), that encodes a peptide,
polypeptide, or protein capable of eliciting an immune response in
a host contacted with the vector. Expression of the polynucleotide
may result in generation of a neutralizing antibody response and/or
a cell mediated response, e.g., a cytotoxic T cell response. In
preferred vector constructs of this invention, the heterologous
polynucleotide is flanked by at least one, preferably two AAV
inverted terminal repeat sequences (ITRs).
[0076] A "helper virus" for AAV refers to a virus that allows AAV
(e.g., wild-type AAV) to be replicated and packaged by a mammalian
cell. A variety of such helper viruses for AAV are known in the
art, including adenoviruses, herpes viruses and poxviruses such as
vaccinia. The adenoviruses encompass a number of different
subgroups, although Adenovirus type 5 of subgroup C is most
commonly used. Numerous adenoviruses of human, non-human mammalian
and avian origin are known and available from depositories such as
the ATCC. Viruses of the herpes family include, for example, herpes
simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as
cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are
also available from depositories such as ATCC.
[0077] An "infectious" virus or viral particle is one that
comprises a polynucleotide component which it is capable of
delivering into a cell for which the viral species is trophic. The
term does not necessarily imply any replication capacity of the
virus. A "replication-competent" virus (e.g., a
replication-competent AAV, sometimes abbreviated as "RCA") refers
to a phenotypically wild-type virus that is infectious, and is also
capable of being replicated in an infected cell (i.e., in the
presence of a helper virus or helper virus functions). In the case
of AAV, replication competence generally requires the presence of
functional AAV packaging genes. Preferred rAAV vectors as described
herein are replication-incompetent in mammalian cells (especially
in human cells) by virtue of the lack of one or more AAV packaging
genes. Preferably, such rAAV vectors lack any AAV packaging gene
sequences in order to minimize the possibility that RCA are
generated by recombination between AAV packaging genes and an
incoming rAAV vector. Preferred rAAV vector preparations as
described herein are those which contain few if any RCA (preferably
less than about 1 RCA per 10.sup.2 rAAV particles, more preferably
less than about 1 RCA per 10.sup.4 rAAV particles, still more
preferably less than about 1 RCA per 10.sup.8 rAAV particles, even
more preferably less than about 1 RCA per 10.sup.12 rAAV particles,
most preferably no RCA).
[0078] The term "polynucleotide" refers to a polymeric form of
nucleotides of any length, including deoxyribonucleotides or
ribonucleotides, or analogs thereof. A polynucleotide may comprise
modified nucleotides, such as methylated or capped nucleotides and
nucleotide analogs, and may be interrupted by non-nucleotide
components. If present, modifications to the nucleotide structure
may be imparted before or after assembly of the polymer. The term
polynucleotide, as used herein, refers interchangeably to double-
and single-stranded molecules. Unless otherwise specified or
required, any embodiment of the invention described herein that is
a polynucleotide encompasses both the double-stranded form and each
of two complementary single-stranded forms known or predicted to
make up the double-stranded form.
[0079] A "transcriptional regulatory sequence" or "TRS," as used
herein, refers to a genomic region that controls the transcription
of a gene or coding sequence to which it is operably linked.
Transcriptional regulatory sequences of use in the present
invention generally include at least one transcriptional promoter
and may also include one or more enhancers and/or terminators of
transcription.
[0080] "Operably linked" refers to an arrangement of two or more
components, wherein the components so described are in a
relationship permitting them to function in a coordinated manner.
By way of illustration, a transcriptional regulatory sequence or a
promoter is operably linked to a coding sequence if the TRS or
promoter promotes transcription of the coding sequence. An operably
linked TRS is generally joined in cis with the coding sequence, but
it is not necessarily directly adjacent to it.
[0081] "Heterologous" means derived from a genotypically distinct
entity from that of the rest of the entity to which it is compared.
For example, a polynucleotide introduced by genetic engineering
techniques into a different cell type is a heterologous
polynucleotide (and, when expressed, can encode a heterologous
polypeptide). Similarly, a TRS or promoter that is removed from its
native coding sequence and operably linked to a different coding
sequence is a heterologous TRS or promoter.
[0082] "Packaging" as used herein refers to a series of subcellular
events that results in the assembly and encapsidation of a viral
vector, particularly an AAV vector. Thus, when a suitable vector is
introduced into a packaging cell line under appropriate conditions,
it can be assembled into a viral particle. Functions associated
with packaging of viral vectors, particularly AAV vectors, are
described herein and in the art.
[0083] A "terminator" refers to a polynucleotide sequence that
tends to diminish or prevent read-through transcription (i.e., it
diminishes or prevent transcription originating on one side of the
terminator from continuing through to the other side of the
terminator). The degree to which transcription is disrupted is
typically a function of the base sequence and/or the length of the
terminator sequence. In particular, as is well known in numerous
molecular biological systems, particular DNA sequences, generally
referred to as "transcriptional termination sequences" are specific
sequences that tend to disrupt read-through transcription by RNA
polymerase, presumably by causing the RNA polymerase molecule to
stop and/or disengage from the DNA being transcribed. Typical
example of such sequence-specific terminators include
polyadenylation ("polyA") sequences, e.g., SV40 polyA. In addition
to or in place of such sequence-specific terminators, insertions of
relatively long DNA sequences between a promoter and a coding
region also tend to disrupt transcription of the coding region,
generally in proportion to the length of the intervening sequence.
This effect presumably arises because there is always some tendency
for an RNA polymerase molecule to become disengaged from the DNA
being transcribed, and increasing the length of the sequence to be
traversed before reaching the coding region would generally
increase the likelihood that disengagement would occur before
transcription of the coding region was completed or possibly even
initiated. Terminators may thus prevent transcription from only one
direction ("uni-directional" terminators) or from both directions
("bi-directional" terminators), and may be comprised of
sequence-specific termination sequences or sequence-non-specific
terminators or both. A variety of such terminator sequences are
known in the art; and illustrative uses of such sequences within
the context of the present invention are provided below.
[0084] "Host cells," "cell lines," "cell cultures," "packaging cell
line" and other such terms denote higher eukaryotic cells,
preferably mammalian cells, most preferably human cells, useful in
the present invention. These cells can be used as recipients for
recombinant vectors, viruses or other transfer polynucleotides, and
include the progeny of the original cell that was transduced. It is
understood that the progeny of a single cell may not necessarily be
completely identical (in morphology or in genomic complement) to
the original parent cell.
[0085] A "therapeutic gene," "prophylactic gene," "target
polynucleotide," "transgene," "gene of interest" and the like
generally refer to a gene or genes to be transferred using a
vector. Typically, in the context of the present invention, such
genes are located within the rAAV vector (which vector is flanked
by inverted terminal repeat (ITR) regions and thus can be
replicated and encapsidated into rAAV particles). Target
polynucleotides can be used in this invention to generate rAAV
vectors for a number of different applications. Such
polynucleotides include, but are not limited to: (i)
polynucleotides encoding proteins useful in other forms of gene
therapy to relieve deficiencies caused by missing, defective or
sub-optimal levels of a structural protein or enzyme; (ii)
polynucleotides that are transcribed into anti-sense molecules;
(iii) polynucleotides that are transcribed into decoys that bind
transcription or translation factors; (iv) polynucleotides that
encode cellular modulators such as cytokines; (v) polynucleotides
that can make recipient cells susceptible to specific drugs, such
as the herpes virus thymidine kinase gene; and (vi) polynucleotides
for cancer therapy, such as E1A tumor suppressor genes or p53 tumor
suppressor genes for the treatment of various cancers. To effect
expression of the transgene in a recipient host cell, it is
preferably operably linked to a promoter, either its own or a
heterologous promoter. A large number of suitable promoters are
known in the art, the choice of which depends on the desired level
of expression of the target polynucleotide; whether one wants
constitutive expression, inducible expression, cell-specific or
tissue-specific expression, etc. The rAAV vector may also contain a
selectable marker.
[0086] A "gene" refers to a polynucleotide containing at least one
open reading frame that is capable of encoding a particular protein
after being transcribed and translated.
[0087] "Recombinant," as applied to a polynucleotide means that the
polynucleotide is the product of various combinations of cloning,
restriction and/or ligation steps, and other procedures that result
in a construct that is distinct from a polynucleotide found in
nature. A recombinant virus is a viral particle comprising a
recombinant polynucleotide. The terms respectively include
replicates of the original polynucleotide construct and progeny of
the original virus construct.
[0088] A "control element" or "control sequence" is a nucleotide
sequence involved in an interaction of molecules that contributes
to the functional regulation of a polynucleotide, including
replication, duplication, transcription, splicing, translation, or
degradation of the polynucleotide. The regulation may affect the
frequency, speed, or specificity of the process, and may be
enhancing or inhibitory in nature. Control elements known in the
art include, for example, transcriptional regulatory sequences such
as promoters and enhancers. A promoter is a DNA region capable
under certain conditions of binding RNA polymerase and initiating
transcription of a coding region usually located downstream (in the
3' direction) from the promoter. Promoters include AAV promoters,
e.g., P5, P19, P40 and AAV ITR promoters, as well as heterologous
promoters.
[0089] An "expression vector" is a vector comprising a region which
encodes a polypeptide of interest, and is used for effecting the
expression of the protein in an intended target cell. An expression
vector also comprises control elements operatively linked to the
encoding region to facilitate expression of the protein in the
target. The combination of control elements and a gene or genes to
which they are operably linked for expression is sometimes referred
to as an "expression cassette," a large number of which are known
and available in the art or can be readily constructed from
components that are available in the art.
[0090] "Genetic alteration" refers to a process wherein a genetic
element is introduced into a cell other than by mitosis or meiosis.
The element may be heterologous to the cell, or it may be an
additional copy or improved version of an element already present
in the cell. Genetic alteration may be effected, for example, by
transfecting a cell with a recombinant plasmid or other
polynucleotide through any process known in the art, such as
electroporation, calcium phosphate precipitation, or contacting
with a polynucleotide-liposome complex. Genetic alteration may also
be effected, for example, by transduction or infection with a DNA
or RNA virus or viral vector. Preferably, the genetic element is
introduced into a chromosome or mini-chromosome in the cell; but
any alteration that changes the phenotype and/or genotype of the
cell and its progeny is included in this term.
[0091] A cell is said to be "stably" altered, transduced or
transformed with a genetic sequence if the sequence is available to
perform its function during extended culture of the cell in vitro.
In preferred examples, such a cell is "inheritably" altered in that
a genetic alteration is introduced which is also inheritable by
progeny of the altered cell.
[0092] The terms "polypeptide" and "protein" are used
interchangeably herein to refer to polymers of amino acids of any
length. The terms also encompass an amino acid polymer that has
been modified; for example, disulfide bond formation,
glycosylation, acetylation, phosphonylation, lipidation, or
conjugation with a labeling component. Polypeptides such as "CFTR"
and the like, when discussed in the context of gene therapy and
compositions therefor, refer to the respective intact polypeptide,
or any fragment or genetically engineered derivative thereof, that
retains the desired biochemical function of the intact protein.
Similarly, references to CFTR, and other such genes for use in gene
therapy (typically referred to as "transgenes" to be delivered to a
recipient cell), include polynucleotides encoding the intact
polypeptide or any fragment or genetically engineered derivative
possessing the desired biochemical function.
[0093] An "isolated" plasmid, virus, or other substance refers to a
preparation of the substance devoid of at least some of the other
components that may also be present where the substance or a
similar substance naturally occurs or is initially prepared from.
Thus, for example, an isolated substance may be prepared by using a
purification technique to enrich it from a source mixture.
Enrichment can be measured on an absolute basis, such as weight per
volume of solution, or it can be measured in relation to a second,
potentially interfering substance present in the source mixture.
Increasing enrichments of the embodiments of this invention are
increasingly more preferred. Thus, for example, a 2-fold enrichment
is preferred, 10-fold enrichment is more preferred, 100-fold
enrichment is more preferred, 1000-fold enrichment is even more
preferred.
[0094] A preparation of AAV is said to be "substantially free" of
helper virus if the ratio of infectious AAV particles to infectious
helper virus particles is at least about 10.sup.2:1; preferably at
least about 10.sup.4:1, more preferably at least about 10.sup.6:1;
still more preferably at least about 10.sup.8:1. Preparations are
also preferably free of equivalent amounts of helper virus proteins
(i.e., proteins as would be present as a result of such a level of
helper virus if the helper virus particle impurities noted above
were present in disrupted form). Viral and/or cellular protein
contamination can generally be observed as the presence of
Coomassie staining bands on SDS gels (e.g., the appearance of bands
other than those corresponding to the AAV capsid proteins VP1, VP2
and VP3).
[0095] "Efficiency" when used in describing viral production,
replication or packaging refers to useful properties of the method:
in particular, the growth rate and the number of virus particles
produced per cell. "High efficiency" production indicates
production of at least 100 viral particles per cell; preferably at
least about 10,000 and more preferably at least about 100,000
particles per cell, over the course of the culture period
specified.
[0096] An "individual" or "subject" treated in accordance with this
invention refers to vertebrates, particularly members of a
mammalian species, and includes but is not limited to domestic
animals, sports animals, and primates, including humans.
[0097] "Treatment" of an individual or a cell is any type of
intervention in an attempt to alter the natural course of the
individual or cell at the time the treatment is initiated, e.g.,
eliciting a prophylactic, curative or other beneficial effect in
the individual. For example, treatment of an individual may be
undertaken to decrease or limit the pathology caused by any
pathological condition, including (but not limited to) an inherited
or induced genetic deficiency, infection by a viral, bacterial, or
parasitic organism, a neoplastic or aplastic condition, or an
immune system dysfunction such as autoimmunity or
immunosuppression. Treatment includes (but is not limited to)
administration of a composition, such as a pharmaceutical
composition, and administration of compatible cells that have been
treated with a composition. Treatment may be performed either
prophylactically or therapeutically; that is, either prior or
subsequent to the initiation of a pathologic event or contact with
an etiologic agent.
[0098] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
virology, microbiology, recombinant DNA, and immunology, which are
within the skill of the art. Such techniques are explained fully in
the literature. See, e.g., Sambrook et al., 1989; Gait, 1984;
Freshney, 1987; the series Methods in Enzymology (Academic Press,
Inc.); Miller et al., 1987; Weir et al., 1996; Ausubel et al.,
1998; Coligan et al., 1991; Coligan et al., 1995; and Scopes
1994.
I. rAAV Vectors
[0099] Recombinant AAV vectors are potentially powerful tools for
human gene therapy, particularly for diseases such as cystic
fibrosis and sickle cell anemia. A major advantage of rAAV vectors
over other approaches to gene therapy is that they generally do not
require ongoing replication of the target cell in order to become
stably integrated into the host cell.
[0100] rAAV vectors and/or viruses are also potentially powerful
for the development of therapeutic or prophylactic vaccines to
prevent infection, progression, and/or severity of disease. A major
advantage of rAAV vectors for vaccine development is that they are
capable of persisting for essentially the lifetime of the cell as a
nuclear episome and therefore provide long term expression of the
peptide, polypeptide, or protein of immunologic interest.
Transgenes of interest include viral gene e.g. the envelope (env)
or gag genes of HIV; bacterial genes e.g., streptococcal cell wall
proteins; fungi, e.g., cocidomycosis; parasites, e.g.,
Leischmaniosis, or cancer genes, e.g. p53.
[0101] rAAV vectors and/or viruses may also contain one or more
detectable markers. A variety of such markers are known, including,
by way of illustration, the bacterial beta-galactosidase (lacZ)
gene; the human placental alkaline phosphatase (AP) gene and genes
encoding various cellular surface markers which have been used as
reporter molecules both in vitro and in vivo. The rAAV vectors
and/or viruses may also contain one or more selectable markers.
[0102] Recombinant AAV vectors and/or viruses can also comprise
polynucleotides that do not encode proteins, including, e.g.,
polynucleotides encoding for antisense mRNA (the complement of
mRNA) which can be used to block the translation of normal mRNA by
forming a duplex with it, and polynucleotides that encode ribozymes
(RNA catalysts).
II. Selection and Preparation of AAV Vector
[0103] Adeno-associated viruses of any serotype are suitable to
prepare rAAV, since the various serotypes are functionally and
structurally related, even at the genetic level (see, e.g.,
Blacklow, 1988; and Rose, 1974). All AAV serotypes apparently
exhibit similar replication properties mediated by homologous rep
genes; and all generally bear three related capsid proteins such as
those expressed in AAV2. The degree of relatedness is further
suggested by heteroduplex analysis which reveals extensive
cross-hybridization between serotypes along the length of the
genome; and the presence of analogous self-annealing segments at
the termini that correspond to ITRs. The similar infectivity
patterns also suggest that the replication functions in each
serotype are under similar regulatory control. Among the various
AAV serotypes, AAV2 is most commonly employed.
[0104] An AAV vector of the invention typically comprises a
polynucleotide that is heterologous to AAV. The polynucleotide is
typically of interest because of a capacity to provide a function
to a target cell in the context of gene therapy, such as up- or
down-regulation of the expression of a certain phenotype. Such a
heterologous polynucleotide or "transgene," generally is of
sufficient length to provide the desired function or encoding
sequence.
[0105] Where transcription of the heterologous polynucleotide is
desired in the intended target cell, it can be operably linked to
its own or to a heterologous promoter, depending for example on the
desired level and/or specificity of transcription within the target
cell, as is known in the art. Various types of promoters and
enhancers are suitable for use in this context. Constitutive
promoters provide an ongoing level of gene transcription, and are
preferred when it is desired that the therapeutic or prophylactic
polynucleotide be expressed on an ongoing basis. Inducible
promoters generally exhibit low activity in the absence of the
inducer, and are up-regulated in the presence of the inducer. They
may be preferred when expression is desired only at certain times
or at certain locations, or when it is desirable to titrate the
level of expression using an inducing agent. Promoters and
enhancers may also be tissue-specific: that is, they exhibit their
activity only in certain cell types, presumably due to gene
regulatory elements found uniquely in those cells.
[0106] Illustrative examples of promoters are the SV40 late
promoter from simian virus 40, the Baculovirus polyhedron
enhancer/promoter element, Herpes Simplex Virus thymidine kinase
(HSV tk), the immediate early promoter from cytomegalovirus (CMV)
and various retroviral promoters including LTR elements. Inducible
promoters include heavy metal ion inducible promoters (such as the
mouse mammary tumor virus (mMTV) promoter or various growth hormone
promoters), and the promoters from T7 phage which are active in the
presence of T7 RNA polymerase. By way of illustration, examples of
tissue-specific promoters include various surfactin promoters (for
expression in the lung), myosin promoters (for expression in
muscle), and albumin promoters (for expression in the liver). A
large variety of other promoters are known and generally available
in the art, and the sequences of many such promoters are available
in sequence databases such as the GenBank database.
[0107] Where translation is also desired in the intended target
cell, the heterologous polynucleotide will preferably also comprise
control elements that facilitate translation (such as a ribosome
binding site or "RBS" and a polyadenylation signal). Accordingly,
the heterologous polynucleotide generally comprises at least one
coding region operatively linked to a suitable promoter, and may
also comprise, for example, an operatively linked enhancer,
ribosome binding site and poly-A signal. The heterologous
polynucleotide may comprise one encoding region, or more than one
encoding regions under the control of the same or different
promoters. The entire unit, containing a combination of control
elements and encoding region, is often referred to as an expression
cassette.
[0108] The heterologous polynucleotide is integrated by recombinant
techniques into or preferably in place of the AAV genomic coding
region (i.e., in place of the AAV rep and cap genes), but is
generally flanked on either side by AAV inverted terminal repeat
(ITR) regions. This means that an ITR appears both upstream and
downstream from the coding sequence, either in direct
juxtaposition, preferably (although not necessarily) without any
intervening sequence of AAV origin in order to reduce the
likelihood of recombination that might regenerate a
replication-competent AAV genome. However, a single ITR may be
sufficient to carry out the functions normally associated with
configurations comprising two ITRs (see, for example, WO 94/13788),
and vector constructs with only one ITR can thus be employed in
conjunction with the packaging and production methods of the
present invention.
[0109] The native promoters for rep are self-regulating, and can
limit the amount of AAV particles produced. The rep gene can also
be operably linked to a heterologous promoter, whether rep is
provided as part of the vector construct, or separately. Any
heterologous promoter that is not strongly down-regulated by rep
gene expression is suitable; but inducible promoters are preferred
because constitutive expression of the rep gene can have a negative
impact on the host cell. A large variety of inducible promoters are
known in the art; including, by way of illustration, heavy metal
ion inducible promoters (such as metallothionein promoters);
steroid hormone inducible promoters (such as the MMTV promoter or
growth hormone promoters); and promoters such as those from T7
phage which are active in the presence of T7 RNA polymerase. An
especially preferred sub-class of inducible promoters are those
that are induced by the helper virus that is used to complement the
replication and packaging of the rAAV vector. A number of
helper-virus-inducible promoters have also been described,
including the adenovirus early gene promoter which is inducible by
adenovirus EIA protein; the adenovirus major late promoter; the
herpesvirus promoter which is inducible by herpesvirus proteins
such as VP16 or 1 CP4; as well as vaccinia or poxvirus inducible
promoters.
[0110] Methods for identifying and testing helper-virus-inducible
promoters have been described (see, e.g., WO 96/17947). Thus,
methods are known in the art to determine whether or not candidate
promoters are helper-virus-inducible, and whether or not they will
be useful in the generation of high efficiency packaging cells.
Briefly, one such method involves replacing the p5 promoter of the
AAV rep gene with the putative helper-virus-inducible promoter
(either known in the art or identified using well-known techniques
such as linkage to promoter-less "reporter" genes). The AAV rep-cap
genes (with p5 replaced), preferably linked to a positive
selectable marker such as an antibiotic resistance gene, are then
stably integrated into a suitable host cell (such as the HeLa or
A549 cells exemplified below). Cells that are able to grow
relatively well under selection conditions (e.g., in the presence
of the antibiotic) are then tested for their ability to express the
rep and cap genes upon addition of a helper virus. As an initial
test for rep and/or cap expression, cells can be readily screened
using immunofluorescence to detect Rep and/or Cap proteins.
Confirmation of packaging capabilities and efficiencies can then be
determined by functional tests for replication and packaging of
incoming rAAV vectors. Using this methodology, a
helper-virus-inducible promoter derived from the mouse
metallothionein gene has been identified as a suitable replacement
for the p5 promoter, and used for producing high titers of rAAV
particles (as described in WO 96/17947).
[0111] Given the relative encapsidation size limits of various AAV
genomes, insertion of a large heterologous polynucleotide into the
genome necessitates removal of a portion of the AAV sequence.
Removal of one or more AAV genes is in any case desirable, to
reduce the likelihood of generating replication-competent AAV
("RCA"). Accordingly, encoding or promoter sequences for rep, cap,
or both, are preferably removed, since the functions provided by
these genes can be provided in trans.
[0112] The resultant vector is referred to as being "defective" in
these functions. In order to replicate and package the vector, the
missing functions are complemented with a packaging gene, or a
plurality thereof, which together encode the necessary functions
for the various missing rep and/or cap gene products. The packaging
genes or gene cassettes are preferably not flanked by AAV ITRs and
preferably do not share any substantial homology with the rAAV
genome. Thus, in order to minimize homologous recombination during
replication between the vector sequence and separately provided
packaging genes, it is desirable to avoid overlap of the two
polynucleotide sequences. The level of homology and corresponding
frequency of recombination increase with increasing length of
homologous sequences and with their level of shared identity. The
level of homology that will pose a concern in a given system can be
determined theoretically and confirmed experimentally, as is known
in the art. Typically, however, recombination can be substantially
reduced or eliminated if the overlapping sequence is less than
about a 25 nucleotide sequence if it is at least 80% identical over
its entire length, or less than about a 50 nucleotide sequence if
it is at least 70% identical over its entire length. Of course,
even lower levels of homology are preferable since they will
further reduce the likelihood of recombination. It appears that,
even without any overlapping homology, there is some residual
frequency of generating RCA. Even further reductions in the
frequency of generating RCA (e.g., by nonhomologous recombination)
can be obtained by "splitting" the replication and encapsidation
functions of AAV, as described by Allen et al., WO 98/27204).
[0113] The rAAV vector construct, and the complementary packaging
gene constructs can be implemented in this invention in a number of
different forms. Viral particles, plasmids, and stably transformed
host cells can all be used to introduce such constructs into the
packaging cell, either transiently or stably.
[0114] In certain embodiments of this invention, the AAV vector and
complementary packaging gene(s), if any, are provided in the form
of bacterial plasmids, AAV particles, or any combination thereof.
In other embodiments, either the AAV vector sequence, the packaging
gene(s), or both, are provided in the form of genetically altered
(preferably inheritably altered) eukaryotic cells. The development
of host cells inheritably altered to express the AAV vector
sequence, AAV packaging genes, or both, provides an established
source of the material that is expressed at a reliable level.
[0115] A variety of different genetically altered cells can thus be
used in the context of this invention. By way of illustration, a
mammalian host cell may be used with at least one intact copy of a
stably integrated rAAV vector. An AAV packaging plasmid comprising
at least an AAV rep gene operably linked to a promoter can be used
to supply replication functions (as described in U.S. Pat. No.
5,658,776). Alternatively, a stable mammalian cell line with an AAV
rep gene operably linked to a promoter can be used to supply
replication functions (see, e.g., Trempe et al., WO 95/13392);
Burstein et al. (WO 98/23018); and Johnson et al. (U.S. Pat. No.
5,656,785). The AAV cap gene, providing the encapsidation proteins
as described above, can be provided together with an AAV rep gene
or separately (see, e.g., the above-referenced applications and
patents as well as Allen et al. (WO 98/27204). Other combinations
are possible and included within the scope of this invention.
III. Generating rAAV
[0116] To generate recombinant AAV particles useful for such
purposes as gene therapy, the packaging cell line is preferably
supplied with a recombinant AAV vector comprising AAV inverted
terminal repeat (ITR) regions surrounding one or more
polynucleotides of interest (or "target" polynucleotides).
[0117] The target polynucleotide is generally operably linked to a
promoter, either its own or a heterologous promoter. A large number
of suitable promoters are known in the art, the choice of which
depends on the desired level of expression of the target
polynucleotide (i.e., whether one wants constitutive expression,
inducible expression, cell-specific or tissue-specific expression,
etc.).
[0118] Preferably, the rAAV vector also contains a positive
selectable marker in order to allow for selection of cells that
have been infected by the rAAV vector. Negative selectable markers
can also be included; as a means of selecting against those same
cells should that become necessary or desirable. In a preferred
embodiment, one can make use of the "bifunctional selectable fusion
genes" described by S. D. Lupton; see, e.g., PCT/US91/08442 and
PCT/US94/05601. Briefly, those constructs involve direct
translational fusions between a dominant positive selectable marker
and a negative selectable marker. Preferred positive selectable
markers are derived from genes selected from the group consisting
of hph, neo, and gpt, and preferred negative selectable markers are
derived from genes selected from the group consisting of cytosine
deaminase, HSV-I TK, VZV TK, HPRT, APRT and gpt. Especially
preferred markers are bifunctional selectable fusion genes wherein
the positive selectable marker is derived from hph or neo, and the
negative selectable marker is derived from cytosine deaminase or a
TK gene.
[0119] Useful target polynucleotides can be employed in rAAV
vectors for a number of different applications. Such
polynucleotides include, but are not limited to: (i)
polynucleotides encoding proteins useful in other forms of gene
therapy to relieve deficiencies caused by missing, defective or
sub-optimal levels of a structural protein or enzyme; (ii)
polynucleotides that are transcribed into anti-sense molecules;
(iii) polynucleotides that are transcribed into decoys that bind
transcription or translation factors; (iv) polynucleotides that
encode cellular modulators such as cytokines; (v) polynucleotides
that can make recipient cells susceptible to specific drugs, such
as the herpes virus thymidine kinase gene; and (vi) polynucleotides
for cancer therapy, such as the wild-type p53 tumor suppressor cDNA
for replacement of the missing or damaged p53 gene associated with
some lung and breast cancers, or the E1A tumor suppressor gene
which is capable of inhibiting tumorigenesis and/or metastasis of a
variety of different cancers including breast and ovarian
cancers.
[0120] Since the therapeutic or prophylactic specificity of the
resulting recombinant AAV particle is determined by the particular
vector or pro-vector introduced, the same basic packaging cell line
can be modified for any of these applications. For example, a
vector comprising a specific target polynucleotide can be
introduced into the packaging cell for production of the AAV vector
by any of several possible methods; including, for example,
electroporation or transfection of a plasmid comprising an rAAV
pro-vector, or infection with an rAAV or helper virus comprising an
rAAV vector or pro-vector.
[0121] Helper virus can be introduced before, during or after
introduction of the rAAV vector. For example, the plasmid can be
co-infected into the culture along with the helper virus; and the
cells can then be cultured for a sufficient period, typically 2-5
days, in conditions suitable for replication and packaging as known
in the art (see references above and examples below). Lysates are
prepared, and the recombinant AAV vector particles are purified by
techniques known in the art.
[0122] In a preferred embodiment, also illustrated in the Examples
below, a recombinant AAV vector is itself stably integrated into a
mammalian cell to be used for packaging. Such rAAV "producer cells"
can then be grown and stored until ready for use. To induce
production of rAAV particles from such producer cells, the user
need only infect the cells with helper virus and culture the cells
under conditions suitable for replication and packaging of AAV (as
described below).
[0123] Alternatively, one or more of the AAV split-packaging genes
or the rAAV vector can be introduced as part of a recombinant
helper virus. For example, the E1, E3 and/or the E4 genes of
adenovirus can be replaced with one or more split-packaging genes
or an rAAV vector. Techniques for facilitating cloning into
adenovirus vectors, e.g., into the E1 and/or E3 regions, are known
in the art (see, e.g., Bett, A. J. et al., Proc. Natl. Acad. Sci.
USA, 91, 8802-8806 (1994)). Thus, a helper virus such as a
recombinant adenovirus, can be used to provide helper virus
functions as well as AAV packaging genes and/or an rAAV pro-vector,
since (as is known in the art) a number of genes in such a helper
virus (e.g., the E3 gene of adenovirus) can be replaced without
eliminating helper virus activity. Additional genes can be inserted
into such a helper virus by providing any necessary helper virus
functions in trans. For example, human 293 cells contain adenoviral
genes that can complement adenoviral E1 mutants. Thus, heterologous
genes can also be cloned into an adenovirus in which the E1 genes
have been deleted, for use in cells that can effectively provide
such adenoviral functions in trans. Alternatively, the use of a
helper virus can be eliminated by providing all necessary helper
virus functions in the packaging cell.
IV. Introduction of Genetic Material Into Cells
[0124] As is described in the art, and illustrated both herein and
in the references cited above, genetic material can be introduced
into cells (such as mammalian "producer" cells for the production
of AAV) using any of a variety of means to transform or transduce
such cells. By way of illustration, such techniques include, for
example, transfection with bacterial plasmids, infection with viral
vectors, electroporation, calcium phosphate precipitation, and
introduction using any of a variety of lipid-based compositions (a
process often referred to as "lipofection"). Methods and
compositions for performing these techniques have been described in
the art and are widely available.
[0125] Selection of suitably altered cells may be conducted by any
technique in the art. For example, the polynucleotide sequences
used to alter the cell may be introduced simultaneously with or
operably linked to one or more detectable or selectable markers as
is known in the art. By way of illustration, one can employ a
drug-resistance gene as a selectable marker. Drug-resistant cells
can then be picked and grown, and then tested for expression of the
desired sequence, i.e., a packaging gene product, or a product of
the heterologous polynucleotide, as appropriate. Testing for
acquisition, localization and/or maintenance of an introduced
polynucleotide can be performed using DNA hybridization-based
techniques (such as Southern blotting and other procedures as is
known in the art). Testing for expression can be readily performed
by Northern analysis of RNA extracted from the genetically altered
cells, or by indirect immunofluorescence for the corresponding gene
product. Testing and confirmation of packaging capabilities and
efficiencies can be obtained by introducing to the cell the
remaining functional components of AAV and a helper virus, to test
for production of AAV particles. Where a cell is inheritably
altered with a plurality of polynucleotide constructs, it is
generally more convenient (though not essential) to introduce them
to the cell separately, and validate each step seriatim. References
describing such techniques include those cited herein.
V. Selection and Preparation of Helper Virus
[0126] As discussed above, AAV is a parvovirus that is defective
for self-replication, and must generally rely on a helper virus to
supply certain replicative functions. A number of such helper
viruses have been identified, including adenoviruses, herpes
viruses (including but not limited to HSV1, cytomegalovirus and
HHV-6), and pox viruses (particularly vaccinia). Any such virus may
be used with this invention.
[0127] Frequently, the helper virus is an adenovirus of a type and
subgroup that can infect the intended host cell. Human adenovirus
of subgroup C, particularly serotypes 1, 2, 4, 6, and 7, are
commonly used. Serotype 5 is generally preferred.
[0128] The features and growth patterns of adenovirus are known in
the art. The reader may refer, for example, to Horowitz (1985). The
packaged adenovirus genome is a linear DNA molecule, linked through
adenovirus ITRs at the left- and right-hand termini through a
terminal protein complex to form a circle. Control and encoding
regions for early, intermediate, and late components overlap within
the genome. Early region genes are implicated in replication of the
adenovirus genome, and are grouped depending on their location into
the E1, E2, E3, and E4 regions.
[0129] Although not essential, in principle it is desirable that
the helper virus strain be defective for replication in the subject
ultimately to receive the genetic therapy. Thus, any residual
helper virus present in an rAAV preparation will be
replication-incompetent. Adenoviruses from which the E1A or both
the E1A and the E3 region have been removed are not infectious for
most human cells. They can be replicated in a permissive cell line
(e.g., the human 293 cell line) which is capable of complementing
the missing activity. Regions of adenovirus that appear to be
associated with helper function, as well as regions that do not,
have been identified and described in the art (see, e.g., P. Colosi
et al., WO97/17458, and references cited therein).
VI. Uses of rAAV for Gene Therapy
[0130] AAV vectors can be used for administration to an individual
for purposes of gene therapy or vaccination. Suitable diseases for
rAAV therapy include but are not limited to those induced by viral,
bacterial, or parasitic infections, various malignancies and
hyperproliferative conditions, autoimmune conditions, and
congenital deficiencies.
[0131] Gene therapy can be conducted to enhance the level of
expression of a particular protein either within or secreted by the
cell. Vectors of this invention may be used to genetically alter
cells either for gene marking, replacement of a missing or
defective gene, or insertion of a therapeutic gene. Alternatively,
a polynucleotide may be provided to the cell that decreases the
level of expression. This may be used for the suppression of an
undesirable phenotype, such as the product of a gene amplified or
overexpressed during the course of a malignancy, or a gene
introduced or overexpressed during the course of a microbial
infection. Expression levels may be decreased by supplying a
therapeutic or prophylactic polynucleotide comprising a sequence
capable, for example, of forming a stable hybrid with either the
target gene or RNA transcript (antisense therapy), capable of
acting as a ribozyme to cleave the relevant mRNA or capable of
acting as a decoy for a product of the target gene.
[0132] The introduction of rAAV vectors by the methods of the
present invention may involve use of any number of delivery
techniques (both surgical and non-surgical) which are available and
well known in the art. Such delivery techniques, for example,
include vascular catheterization, cannulization, injection,
inhalation, endotracheal, subcutaneous, inunction, topical, oral,
percutaneous, intra-arterial, intravenous, and/or intraperitoneal
administrations. Vectors can also be introduced by way of
bioprostheses, including, by way of illustration, vascular grafts
(PTFE and dacron), heart valves, intravascular stents,
intravascular paving as well as other non-vascular prostheses.
General techniques regarding delivery, frequency, composition and
dosage ranges of vector solutions are within the skill of the
art.
[0133] In particular, for delivery of a vector of the invention to
a tissue, any physical or biological method that will introduce the
vector to a host animal can be employed. Vector means both a bare
recombinant vector and vector DNA packaged into viral coat
proteins, as is well known for AAV administration. Simply
dissolving an AAV vector in phosphate buffered saline has been
demonstrated to be sufficient to provide a vehicle useful for
muscle tissue expression, and there are no known restrictions on
the carriers or other components that can be coadministered with
the vector (although compositions that degrade DNA should be
avoided in the normal manner with vectors). Pharmaceutical
compositions can be prepared as injectable formulations or as
topical formulations to be delivered to the muscles by transdermal
transport. Numerous formulations for both intramuscular injection
and transdermal transport have been previously developed and can be
used in the practice of the invention. The vectors can be used with
any pharmaceutically acceptable carrier for ease of administration
and handling.
[0134] For purposes of intramuscular injection, solutions in an
adjuvant such as sesame or peanut oil or in aqueous propylene
glycol can be employed, as well as sterile aqueous solutions. Such
aqueous solutions can be buffered, if desired, and the liquid
diluent first rendered isotonic with saline or glucose. Solutions
of the AAV vector as a free acid (DNA contains acidic phosphate
groups) or a pharmacologically acceptable salt can be prepared in
water suitably mixed with a surfactant such as
hydroxypropylcellulose. A dispersion of AAV viral particles can
also be prepared in glycerol, liquid polyethylene glycols and
mixtures thereof and in oils. Under ordinary conditions of storage
and use, these preparations contain a preservative to prevent the
growth of microorganisms. In this connection, the sterile aqueous
media employed are all readily obtainable by standard techniques
well-known to those skilled in the art.
[0135] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases the form must be sterile and must be
fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, liquid polyethylene glycol and
the like), suitable mixtures thereof, and vegetable oils. The
proper fluidity can be maintained, for example, by the use of a
coating such as lecithin, by the maintenance of the required
particle size in the case of a dispersion and by the use of
surfactants. The prevention of the action of microorganisms can be
brought about by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal
and the like. In many cases it will be preferable to include
isotonic agents, for example, sugars or sodium chloride. Prolonged
absorption of the injectable compositions can be brought about by
use of agents delaying absorption, for example, aluminum
monostearate and gelatin.
[0136] Sterile injectable solutions are prepared by incorporating
the AAV vector in the required amount in the appropriate solvent
with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the sterilized active
ingredient into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and the freeze drying technique
which yield a powder of the active ingredient plus any additional
desired ingredient from the previously sterile-filtered solution
thereof.
[0137] For purposes of topical administration, dilute sterile,
aqueous solutions (usually in about 0.1% to 5% concentration),
otherwise similar to the above parenteral solutions, are prepared
in containers suitable for incorporation into a transdermal patch,
and can include known carriers, such as pharmaceutical grade
dimethylsulfoxide (DMSO).
[0138] Of particular interest is the correction of the genetic
defect of cystic fibrosis, by supplying a properly functioning
cystic fibrosis transmembrane conductance regulator (CFTR) to the
airway epithelium. Thus, rAAV vectors encoding native CFTR protein,
and mutants and fragments thereof, are all preferred embodiments of
this invention.
[0139] Compositions of this invention may be used in vivo as well
as ex vivo. In vivo gene therapy comprises administering the
vectors of this invention directly to a subject. Pharmaceutical
compositions can be supplied as liquid solutions or suspensions, as
emulsions, or as solid forms suitable for dissolution or suspension
in liquid prior to use. For administration into the respiratory
tract, a preferred mode of administration is by aerosol, using a
composition that provides either a solid or liquid aerosol when
used with an appropriate aerosolubilizer device. Another preferred
mode of administration into the respiratory tract is using a
flexible fiberoptic bronchoscope to instill the vectors. Typically,
the viral vectors are in a pharmaceutically suitable pyrogen-free
buffer such as Ringer's balanced salt solution (pH 7.4). Although
not required, pharmaceutical compositions may optionally be
supplied in unit dosage form suitable for administration of a
precise amount.
[0140] An effective amount of virus is administered, depending on
the objectives of treatment. An effective amount may be given in
single or divided doses. Where a low percentage of transduction can
cure a genetic deficiency, then the objective of treatment is
generally to meet or exceed this level of transduction. In some
instances, this level of transduction can be achieved by
transduction of only about 1 to 5% of the target cells, but is more
typically 20% of the cells of the desired tissue type, usually at
least about 50%, preferably at least about 80%, more preferably at
least about 95%, and even more preferably at least about 99% of the
cells of the desired tissue type. As a guide, the number of vector
particles present in a single dose given by bronchoscopy will
generally be at least about 1.times.10.sup.8, and is more typically
5.times.10.sup.8, 1.times.10.sup.10 and on some occasions
1.times.10.sup.11 particles, including both DNAse-resistant and
DNAse-susceptible particles. In terms of DNAse-resistant particles,
the dose will generally be between 1.times.10.sup.6 and
1.times.10.sup.14 particles, more generally between about
1.times.10.sup.8 and 1.times.10.sup.12 particles. The treatment can
be repeated as often as every two or three weeks, as required,
although treatment once in 180 days may be sufficient.
[0141] To confirm the presence of the desired DNA sequence in the
host cell, a variety of assays may be performed. Such assays
include, for example, "molecular biological" assays well known to
those of skill in the art, such as Southern and Northern blotting,
RT-PCR and PCR; "biochemical" assays, such as detecting the
presence of a polypeptide expressed from a gene present in the
vector, e.g., by immunological means (immunoprecipitations,
immunoaffinity columns, ELISAs and Western blots) or by any other
assay useful to identify the presence and/or expression of a
particular nucleic acid molecule falling within the scope of the
invention.
[0142] To detect and quantitate RNA produced from introduced DNA
segments, RT-PCR may be employed. In this application of PCR, it is
first necessary to reverse transcribe RNA into DNA, using enzymes
such as reverse transcriptase, and then through the use of
conventional PCR techniques amplify the DNA. In most instances PCR
techniques, while useful, will not demonstrate integrity of the RNA
product. Further information about the nature of the RNA product
may be obtained by Northern blotting. This technique demonstrates
the presence of an RNA species and gives information about the
integrity of that RNA. The presence or absence of an RNA species
can also be determined using dot or slot blot Northern
hybridizations. These techniques are modifications of Northern
blotting and only demonstrate the presence or absence of an RNA
species.
[0143] While Southern blotting and PCR may be used to detect the
DNA segment in question, they do not provide information as to
whether the DNA segment is being expressed. Expression may be
evaluated by specifically identifying the polypeptide products of
the introduced DNA sequences or evaluating the phenotypic changes
brought about by the expression of the introduced DNA segment in
the host cell.
[0144] Thus, the effectiveness of the genetic alteration can be
monitored by several criteria, including analysis of physiological
fluid samples, e.g., urine, plasma, serum, blood, cerebrospinal
fluid or nasal or lung washes. Samples removed by biopsy or
surgical excision may be analyzed by in situ hybridization, PCR
amplification using vector-specific probes, RNAse protection,
immunohistology, or immunofluorescent cell counting. When the
vector is administered by bronchoscopy, lung function tests may be
performed, and bronchial lavage may be assessed for the presence of
inflammatory cytokines. The treated subject may also be monitored
for clinical features, and to determine whether the cells express
the function intended to be conveyed by the therapeutic or
prophylactic polynucleotide.
[0145] The decision of whether to use in vivo or ex vivo therapy,
and the selection of a particular composition, dose, and route of
administration will depend on a number of different factors,
including but not limited to features of the condition and the
subject being treated. The assessment of such features and the
design of an appropriate therapeutic or prophylactic regimen is
ultimately the responsibility of the prescribing physician.
[0146] The foregoing description provides, inter alia, methods for
generating high titer preparations of recombinant AAV vectors that
are substantially free of helper virus (e.g., adenovirus) and
cellular proteins. It is understood that variations may be applied
to these methods by those of skill in this art without departing
from the spirit of this invention.
VII. Exemplary Methods to Identify Useful Agents
[0147] The utility of rAAV as a gene therapy vector is based on its
transduction properties. Methods to detect AAV transduction are
known in the art, including those described herein, and are useful
to screen libraries, types and classes of agents for the ability to
improve AAV transduction by means other than by affecting binding
to cell surface receptors, or the rate of intra-nuclear genome
conversion of the single stranded rAAV vector to a double stranded
genome.
[0148] Transduction may be defined by protein expression of a
heterologous transgene contained in the vector or steady state
levels thereof. Hence, transduction is a measurable functional
endpoint of successful gene delivery with a viral vector, e.g.,
rAAV. A variety of transgenes have been expressed from cells
infected with AAV vectors, and include intracellularly expressed
proteins such as the green fluorescent protein (GFP), cell membrane
associated proteins such as the cystic fibrosis transmembrane
protein (CFTR), and secretory proteins such as Epo, FVIII, and FIX.
However, not all transgenes are capable of fully assessing the
extent of transduction with a given vector and tissue target. For
example, secreted proteins do not give indication of the number of
cell types expressing a given transgene. Furthermore, functional
markers of gene expression are dependent on the ability of a given
transgene protein product to function properly within a given
target cell type.
VIII. Agents Useful in the Practice of the Invention
[0149] rAAV must undergo a number of complex intracellular events
between binding and conversion to dsDNA that may be rate limiting
for transduction efficiency including but not limited to rAAV
endocytosis, trafficking and processing of the rAAV through the
appropriate intracellular compartments (including without
limitation proteosomes, endosomes, and trans-golgi), transport into
the nucleus, and viral uncoating. Furthermore, agents that alter
the efficiency of these intracellular processing events can also
have an end result of increasing the amount of viral DNA in the
nucleus and hence, through steady state, the abundance of gene
conversion products. Thus, an increase in genome conversion
products following enhancement of rAAV intracellular processing
does not necessarily indicate an increased rate of gene conversion.
Methods of enhancing transduction with rAAV are expected to
increase the extent of double stranded genome conversion products
in the nucleus. This is an important distinction with previous
methods aimed at directly enhancing genome conversion using DNA
damaging agents, topoisomerase inhibitors, or adenoviral early gene
products (Alexander et al., 1997; Alexander et al., 1996; Ferrari
et al., 1996; Fisher et al., 1996; Halbert et al., 1997; Russel et
al., 1995) which essentially change the level of gene conversion
enzymes in the nucleus. However, in cases where gene conversion in
the nucleus is not rate limiting, intracellular viral processing
events that limit transduction may predominate.
[0150] Thus, agents useful in the practice of the invention include
agents which alter rAAV transduction efficiency, e.g., rAAV
endocytosis, trafficking and processing of rAAV through the
intracellular compartment, viral nucleic acid or protein
degradation, viral uncoating and nuclear transport of virus or
viral genomes or otherwise modulate proteosomes. Preferred agents
are those which enhance or increase rAAV transduction. Classes of
agents useful in the invention include but are not limited to
antibiotics, chemotherapeutics, e.g., anthracyclines, proteosome
modulators, lipid lowering agents, and food additives. Exemplary
agents include proteasomes (Wagner et al., 2002; Young et al.,
2000; Seisenberger et al., 2001), as well as agents that modulate
the proteosome and ubiquitin pathways, e.g., bind to proteosomes
and/or modulate the activity of proteosomes, ubiquitin, ubiquitin
carrier protein, or ubiquitin ligase, but do not substantially
alter the activity of the proteosome, e.g., the proteolytic
activity of the proteasome or of ubiquitin, ubiquitin carrier
protein, or ubiquitin ligase. Examples of these agents include
without limitation antibiotics, e.g., epoxomicin, lipid lowering
drugs, e.g., simvastatin, food additives, e.g., tannic acid, and
chemotherapeutics, e.g., cisplatin, anthracyclines such as
doxorubicin, and camptothecin.
IX. Dosages, Formulations and Routes of Administration of the
Agents of the Invention
[0151] Administration of the agents identified in accordance with
the present invention may be continuous or intermittent, depending,
for example, upon the recipient's physiological condition, whether
the purpose of the administration is therapeutic or prophylactic,
and other factors known to skilled practitioners. The
administration of the agents of the invention may be essentially
continuous over a preselected period of time or may be in a series
of spaced doses. Both local and systemic administration is
contemplated. When the agents of the invention are employed for
prophylactic purposes, agents of the invention are amenable to
chronic use, preferably by systemic administration.
[0152] One or more suitable unit dosage forms comprising the agents
of the invention, which, as discussed below, may optionally be
formulated for sustained release, can be administered by a variety
of routes including oral, or parenteral, including by rectal,
transdermal, subcutaneous, intravenous, intramuscular,
intraperitoneal, intrathoracic, intrapulmonary and intranasal
routes. For example, for administration to the liver, intravenous
administration is preferred. For administration to the lung, airway
administration is preferred. The formulations may, where
appropriate, be conveniently presented in discrete unit dosage
forms and may be prepared by any of the methods well known to
pharmacy. Such methods may include the step of bringing into
association the agent with liquid carriers, solid matrices,
semi-solid carriers, finely divided solid carriers or combinations
thereof, and then, if necessary, introducing or shaping the product
into the desired delivery system.
[0153] When the agents of the invention are prepared for oral
administration, they are preferably combined with a
pharmaceutically acceptable carrier, diluent or excipient to form a
pharmaceutical formulation, or unit dosage form. The total active
ingredients in such formulations comprise from 0.1 to 99.9% by
weight of the formulation. By "pharmaceutically acceptable" it is
meant the carrier, diluent, excipient, and/or salt must be
compatible with the other ingredients of the formulation, and not
deleterious to the recipient thereof. The active ingredient for
oral administration may be present as a powder or as granules; as a
solution, a suspension or an emulsion; or in achievable base such
as a synthetic resin for ingestion of the active ingredients from a
chewing gum. The active ingredient may also be presented as a
bolus, electuary or paste.
[0154] Pharmaceutical formulations containing the agents of the
invention can be prepared by procedures known in the art using well
known and readily available ingredients. For example, the agent can
be formulated with common excipients, diluents, or carriers, and
formed into tablets, capsules, suspensions, powders, and the like.
Examples of excipients, diluents, and carriers that are suitable
for such formulations include the following fillers and extenders
such as starch, sugars, mannitol, and silicic derivatives; binding
agents such as carboxymethyl cellulose, HPMC and other cellulose
derivatives, alginates, gelatin, and polyvinyl-pyrrolidone;
moisturizing agents such as glycerol; disintegrating agents such as
calcium carbonate and sodium bicarbonate; agents for retarding
dissolution such as paraffin; resorption accelerators such as
quaternary ammonium compounds; surface active agents such as cetyl
alcohol, glycerol monostearate; adsorptive carriers such as kaolin
and bentonite; and lubricants such as talc, calcium and magnesium
stearate, and solid polyethyl glycols.
[0155] For example, tablets or caplets containing the agents of the
invention can include buffering agents such as calcium carbonate,
magnesium oxide and magnesium carbonate. Caplets and tablets can
also include inactive ingredients such as cellulose, pregelatinized
starch, silicon dioxide, hydroxy propyl methyl cellulose, magnesium
stearate, microcrystalline cellulose, starch, talc, titanium
dioxide, benzoic acid, citric acid, corn starch, mineral oil,
polypropylene glycol, sodium phosphate, and zinc stearate, and the
like. Hard or soft gelatin capsules containing an agent of the
invention can contain inactive ingredients such as gelatin,
microcrystalline cellulose, sodium lauryl sulfate, starch, talc,
and titanium dioxide, and the like, as well as liquid vehicles such
as polyethylene glycols (PEGs) and vegetable oil. Moreover, enteric
coated caplets or tablets of an agent of the invention are designed
to resist disintegration in the stomach and dissolve in the more
neutral to alkaline environment of the duodenum.
[0156] The agents of the invention can also be formulated as
elixirs or solutions for convenient oral administration or as
solutions appropriate for parenteral administration, for instance
by intramuscular, subcutaneous or intravenous routes.
[0157] The pharmaceutical formulations of the agents of the
invention can also take the form of an aqueous or anhydrous
solution or dispersion, or alternatively the form of an emulsion or
suspension.
[0158] Thus, the therapeutic agent may be formulated for parenteral
administration (e.g., by injection, for example, bolus injection or
continuous infusion) and may be presented in unit dose form in
ampules, pre-filled syringes, small volume infusion containers or
in multi-dose containers with an added preservative. The active
ingredients may take such forms as suspensions, solutions, or
emulsions in oily or aqueous vehicles, and may contain formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the active ingredients may be in powder form,
obtained by aseptic isolation of sterile solid or by lyophilization
from solution, for constitution with a suitable vehicle, e.g.,
sterile, pyrogen-free water, before use.
[0159] These formulations can contain pharmaceutically acceptable
vehicles and adjuvants which are well known in the prior art. It is
possible, for example, to prepare solutions using one or more
organic solvent(s) that is/are acceptable from the physiological
standpoint, chosen, in addition to water, from solvents such as
acetone, ethanol, isopropyl alcohol, glycol ethers such as the
products sold under the name "Dowanol", polyglycols and
polyethylene glycols, C.sub.1-C.sub.4 alkyl esters of short-chain
acids, preferably ethyl or isopropyl lactate, fatty acid
triglycerides such as the products marketed under the name
"Miglyol", isopropyl myristate, animal, mineral and vegetable oils
and polysiloxanes.
[0160] The compositions according to the invention can also contain
thickening agents such as cellulose and/or cellulose derivatives.
They can also contain gums such as xanthan, guar or carbo gum or
gum arabic, or alternatively polyethylene glycols, bentones and
montmorillonites, and the like.
[0161] It is possible to add, if necessary, an adjuvant chosen from
antioxidants, surfactants, other preservatives, film-forming,
keratolytic or comedolytic agents, perfumes and colorings. Also,
other active ingredients may be added, whether for the conditions
described or some other condition.
[0162] For example, among antioxidants, t-butylhydroquinone,
butylated hydroxyanisole, butylated hydroxytoluene and
.alpha.-tocopherol and its derivatives may be mentioned. The
galenical forms chiefly conditioned for topical application take
the form of creams, milks, gels, dispersion or microemulsions,
lotions thickened to a greater or lesser extent, impregnated pads,
ointments or sticks, or alternatively the form of aerosol
formulations in spray or foam form or alternatively in the form of
a cake of soap.
[0163] Additionally, the agents are well suited to formulation as
sustained release dosage forms and the like. The formulations can
be so constituted that they release the active ingredient only or
preferably in a particular part of the intestinal or respiratory
tract, possibly over a period of time. The coatings, envelopes, and
protective matrices may be made, for example, from polymeric
substances, such as polylactide-glycolates, liposomes,
microemulsions, microparticles, nanoparticles, or waxes. These
coatings, envelopes, and protective matrices are useful to coat
indwelling devices, e.g., stents, catheters, peritoneal dialysis
tubing, and the like.
[0164] The agents of the invention can be delivered via patches for
transdermal administration. See U.S. Pat. No. 5,560,922 for
examples of patches suitable for transdermal delivery of an agent.
Patches for transdermal delivery can comprise a backing layer and a
polymer matrix which has dispersed or dissolved therein an agent,
along with one or more skin permeation enhancers. The backing layer
can be made of any suitable material which is impermeable to the
agent. The backing layer serves as a protective cover for the
matrix layer and provides also a support function. The backing can
be formed so that it is essentially the same size layer as the
polymer matrix or it can be of larger dimension so that it can
extend beyond the side of the polymer matrix or overlay the side or
sides of the polymer matrix and then can extend outwardly in a
manner that the surface of the extension of the backing layer can
be the base for an adhesive means. Alternatively, the polymer
matrix can contain, or be formulated of, an adhesive polymer, such
as polyacrylate or acrylate/vinyl acetate copolymer. For long-term
applications it might be desirable to use microporous and/or
breathable backing laminates, so hydration or maceration of the
skin can be minimized.
[0165] Examples of materials suitable for making the backing layer
are films of high and low density polyethylene, polypropylene,
polyurethane, polyvinylchloride, polyesters such as poly(ethylene
phthalate), metal foils, metal foil laminates of such suitable
polymer films, and the like. Preferably, the materials used for the
backing layer are laminates of such polymer films with a metal foil
such as aluminum foil. In such laminates, a polymer film of the
laminate will usually be in contact with the adhesive polymer
matrix.
[0166] The backing layer can be any appropriate thickness which
will provide the desired protective and support functions. A
suitable thickness will be from about 10 to about 200 microns.
[0167] Generally, those polymers used to form the biologically
acceptable adhesive polymer layer are those capable of forming
shaped bodies, thin walls or coatings through which agents can pass
at a controlled rate. Suitable polymers are biologically and
pharmaceutically compatible, nonallergenic and insoluble in and
compatible with body fluids or tissues with which the device is
contacted. The use of soluble polymers is to be avoided since
dissolution or erosion of the matrix by skin moisture would affect
the release rate of the agents as well as the capability of the
dosage unit to remain in place for convenience of removal.
[0168] Exemplary materials for fabricating the adhesive polymer
layer include polyethylene, polypropylene, polyurethane,
ethylene/propylene copolymers, ethylene/ethylacrylate copolymers,
ethylene/vinyl acetate copolymers, silicone elastomers, especially
the medical-grade polydimethylsiloxanes, neoprene rubber,
polyisobutylene, polyacrylates, chlorinated polyethylene, polyvinyl
chloride, vinyl chloride-vinyl acetate copolymer, crosslinked
polymethacrylate polymers (hydrogel), polyvinylidene chloride,
poly(ethylene terephthalate), butyl rubber, epichlorohydrin
rubbers, ethylene vinyl alcohol copolymers,
ethylene-vinyloxyethanol copolymers; silicone copolymers, for
example, polysiloxane-polycarbonate copolymers,
polysiloxane-polyethylene oxide copolymers,
polysiloxane-polymethacrylate copolymers, polysiloxane-alkylene
copolymers (e.g., polysiloxane-ethylene copolymers),
polysiloxane-alkylenesilane copolymers (e.g.,
polysiloxane-ethylenesilane copolymers), and the like; cellulose
polymers, for example methyl or ethyl cellulose, hydroxy propyl
methyl cellulose, and cellulose esters; polycarbonates;
polytetrafluoroethylene; and the like.
[0169] Preferably, a biologically acceptable adhesive polymer
matrix should be selected from polymers with glass transition
temperatures below room temperature. The polymer may, but need not
necessarily, have a degree of crystallinity at room temperature.
Cross-linking monomeric units or sites can be incorporated into
such polymers. For example, cross-linking monomers can be
incorporated into polyacrylate polymers, which provide sites for
cross-linking the matrix after dispersing the agent into the
polymer. Known cross-linking monomers for polyacrylate polymers
include polymethacrylic esters of polyols such as butylene
diacrylate and dimethacrylate, trimethylol propane trimethacrylate
and the like. Other monomers which provide such sites include allyl
acrylate, allyl methacrylate, diallyl maleate and the like.
[0170] Preferably, a plasticizer and/or humectant is dispersed
within the adhesive polymer matrix. Water-soluble polyols are
generally suitable for this purpose. Incorporation of a humectant
in the formulation allows the dosage unit to absorb moisture on the
surface of skin which in turn helps to reduce skin irritation and
to prevent the adhesive polymer layer of the delivery system from
failing.
[0171] Agents released from a transdermal delivery system must be
capable of penetrating each layer of skin. In order to increase the
rate of permeation of an agent, a transdermal drug delivery system
must be able in particular to increase the permeability of the
outermost layer of skin, the stratum corneum, which provides the
most resistance to the penetration of molecules. The fabrication of
patches for transdermal delivery of agents is well known to the
art.
[0172] For administration to the upper (nasal) or lower respiratory
tract by inhalation, the agents of the invention are conveniently
delivered from an insufflator, nebulizer or a pressurized pack or
other convenient means of delivering an aerosol spray. Pressurized
packs may comprise a suitable propellant such as
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol, the dosage unit may be
determined by providing a valve to deliver a metered amount.
[0173] Alternatively, for administration by inhalation or
insufflation, the composition may take the form of a dry powder,
for example, a powder mix of the agent and a suitable powder base
such as lactose or starch. The powder composition may be presented
in unit dosage form in, for example, capsules or cartridges, or,
e.g., gelatine or blister packs from which the powder may be
administered with the aid of an inhalator, insufflator or a
metered-dose inhaler.
[0174] For intra-nasal administration, the agent may be
administered via nose drops, a liquid spray, such as via a plastic
bottle atomizer or metered-dose inhaler. Typical of atomizers are
the Mistometer (Wintrop) and the Medihaler (Riker).
[0175] The local delivery of the agents of the invention can also
be by a variety of techniques which administer the agent at or near
the site of disease. Examples of site-specific or targeted local
delivery techniques are not intended to be limiting but to be
illustrative of the techniques available. Examples include local
delivery catheters, such as an infusion or indwelling catheter,
e.g., a needle infusion catheter, shunts and stents or other
implantable devices, site specific carriers, direct injection, or
direct applications.
[0176] For topical administration, the agents may be formulated as
is known in the art for direct application to a target area.
Conventional forms for this purpose include wound dressings, coated
bandages or other polymer coverings, ointments, creams, lotions,
pastes, jellies, sprays, and aerosols. Ointments and creams may,
for example, be formulated with an aqueous or oily base with the
addition of suitable thickening and/or gelling agents. Lotions may
be formulated with an aqueous or oily base and will in general also
contain one or more emulsifying agents, stabilizing agents,
dispersing agents, suspending agents, thickening agents, or
coloring agents. The active ingredients can also be delivered via
iontophoresis, e.g., as disclosed in U.S. Pat. Nos. 4,140,122;
4,383,529; or 4,051,842. The percent by weight of an agent of the
invention present in a topical formulation will depend on various
factors, but generally will be from 0.01% to 95% of the total
weight of the formulation, and typically 0.1-25% by weight.
[0177] Drops, such as eye drops or nose drops, may be formulated
with an aqueous or non-aqueous base also comprising one or more
dispersing agents, solubilizing agents or suspending agents. Liquid
sprays are conveniently delivered from pressurized packs. Drops can
be delivered via a simple eye dropper-capped bottle, or via a
plastic bottle adapted to deliver liquid contents dropwise, via a
specially shaped closure.
[0178] The agent may further be formulated for topical
administration in the mouth or throat. For example, the active
ingredients may be formulated as a lozenge further comprising a
flavored base, usually sucrose and acacia or tragacanth; pastilles
comprising the composition in an inert base such as gelatin and
glycerin or sucrose and acacia; and mouthwashes comprising the
composition of the present invention in a suitable liquid
carrier.
[0179] The formulations and compositions described herein may also
contain other ingredients such as antimicrobial agents, or
preservatives. Furthermore, the active ingredients may also be used
in combination with other agents, for example, bronchodilators.
[0180] The agents of this invention may be administered to a mammal
alone or in combination with pharmaceutically acceptable carriers.
As noted above, the relative proportions of active ingredient and
carrier are determined by the solubility and chemical nature of the
compound, chosen route of administration and standard
pharmaceutical practice.
[0181] The dosage of the present agents will vary with the form of
administration, the particular compound chosen and the
physiological characteristics of the particular patient under
treatment. Generally, small dosages will be used initially and, if
necessary, will be increased by small increments until the optimum
effect under the circumstances is reached.
[0182] The invention will be further described by, but is not
limited to, the following examples. In particular, the following
Examples are provided to exemplify various methods to detect rAAV
transduction, which methods are described in WO 00/75365.
EXAMPLE 1
Endosomal Processing Inhibitors May Increase rAAV
Transduction in Polarized Airway Cells
Materials and Methods
[0183] Primary culture of human bronchial epithelia and reagents
utilized. Primary human airway epithelial cells were collected by
enzymatic digestion of bronchial samples from lung transplants, as
previously described (Kondo et al., 1991; Zabner et al., 1996).
Isolated primary airway cells were seeded at a density of
5.times.10.sup.5 cells/cm onto collagen-coated Millicell-HA culture
inserts (Millipore Corp., Bedford, Mass.). Primary cultures were
grown at the air-liquid interface for more than 2 weeks, by which
time differentiation into a mucociliary epithelium occurs. The
culture medium, used to feed only the basolateral side of the
cells, contained 49% DMEM, 49% Ham's F12 and 2% Ultraser G
(BioSepra, Cedex, France). Dimethyl Sulphoxide (DMSO), camptothecin
(Camp), etoposide (Etop), aphidicolin (Aphi), hydroxyurea (HU) and
genistein (Geni) were purchased from Sigma (St. Louis, Mo.).
Tripeptidyl aldehyde proteasome inhibitors
N-Acetyl-L-Leucyl-L-Leucyl-Norleucine (LLnL) and
benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucinal (Z-LLL) were
purchased from Calbiochem-Novabiochem Corporation (La Jolla,
Calif.). Ubiquitin ligase (E3) inhibitors were obtained from Bachem
Bioscience Inc. (King of Prussia, Pa.). Anti-AAV capsid monoclonal
antibody (Anti-VP1, 2 and 3) was purchased from American Research
Products (Belmont, Mass.) and anti-ubiquitin antibody was purchased
from Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.).
[0184] Production of recombinant AAV viral stocks. Recombinant AAV
was produced by a CaPO.sub.4 co-transfection protocol and purified
through three rounds of isopycnic cesium chloride
ultracentrifugation. The proviral plasmid pCisAV.GFP3ori is
described in Duan et al. (1998). The proviral plasmid
pCisRSV.Alkphos, which encodes the alkaline phosphatase reporter
gene under the transcriptional control of the RSV promoter and SV40
poly-adenylation signal, was used to generate AV.Alkphos (Yang et
al., 1999). The proviral plasmid pCisRSV.LacZ used for AV.LacZ
production was generated by first inserting 3474 bp Not I digested
.beta.-galactosidase gene (from pCMV.beta., Clontech) into the Not
I site of the pRep4 (Invitrogene). The entire .beta.-galactosidase
expression cassette, including the RSV promoter,
.beta.-galactosidase reporter gene and SV40 polyA signal, was
excised by Sal I and subsequently cloned into the pSub201 backbone
by blunt end ligation (Samulski et al., 1987). Recombinant viral
stocks were heated at 58.degree. C. for 60 minutes to inactivate
contaminating helper adenovirus. Typical yields were
5.times.10.sup.5 to 5.times.10.sup.9 particles/.mu.l based on DNA
slot blot hybridization assays against plasmid standards. The level
of adenoviral contamination, as based on a second reporter assay
(Duan et al., 1997) for the recombinant adenovirus used for
propagation (Ad.CMVAlkphos for AV.GFP3ori, and Ad.CMVLacZ for
AV.Alkphos, Ad.CMVGFP for AV.LacZ), was less than one functional
particle per 1.times.10.sup.10 rAAV particles used for infection of
293 cells in the presence of adenovirus. Transfection with Rep/Cap
encoding plasmids served as controls for antibody staining of Rep
protein. Virus was dialyzed in PBS prior to in vitro or in vivo
infections.
[0185] Transduction of polarized airway epithelial cells and
primary human fibroblasts. rAAV infection of fully differentiated
bronchial cells was performed as described in Duan et al. (1998).
For infections from the apical surface of the airway cells, 5 .mu.l
rAAV was mixed with 50 .mu.l of culture media and applied directly
onto the apical compartment of Millicell inserts (MOI=10,000
particles/cell). During apical infection, the basolateral side of
the Millicell was continuously bathed in culture media. Gene
transfer to the basal side was performed by inverting Millicell
inserts and applying viral vector to the bottom of the supporting
filter membrane in a 50 .mu.l volume for 2 hours. Subsequently,
Millicell inserts were returned to the upright position, in the
continued presence of the original viral inoculum plus an
additional 450 .mu.l of media. For both apical and basolateral
infections, rAAV containing media was removed after 24 hours and
replaced with either fresh culture media (for the basal side) or
exposed to air (for the apical side). To test the effect of
different agents on the efficiency of AAV transduction in polarized
airway cells, 1 .mu.l of each solution was mixed with AAV prior to
infection of airway epithelia. Agents were usually presented during
the 24 hours AAV infection period unless indicated otherwise. Most
of the agents were dissolved in DMSO except for hydroxyurea
(dissolved in phosphate buffered saline), H-Leu-Ala-OH (dissolved
in 0.9% glacial acetic acid) and H-His-Ala-OH (dissolved in 50%
methanol). The working concentrations of the agents were as
follows: 0.1 .mu.M camptothecin, 10 .mu.M etoposide, 5 .mu.g/ml
aphidicolin, 40 mM hydroxyurea, 50 .mu.M genistein, 40 .mu.M LLnL
and 4 .mu.M Z-LLL. When the ubiquitin ligase (E3) inhibitors
(H-Leu-Ala-OH and H-His-Ala-OH) were used, airway cells were
pretreated with a combination of both inhibitors at a final
concentration of 2 mM for 60 minutes prior to infection, followed
by the continued presence of inhibitor (0.2 mM) during the entire
24 hours infection period from the basolateral surface. Studies
involving EGTA treatment were performed by transiently treating the
apical membrane of polarized airway epithelia with 3 mM EGTA in
water for 10 minutes (Duan et al., 1998). Following hypotonic EGTA
treatment, cultures were washed twice with culture medium and
infected with rAAV in the presence or absence of 40 .mu.M LLnL.
Human primary fibroblast cells (P4) were maintained in 10% fetal
bovine serum (FBS), 1% penicillin and streptomycin, and 89% DMEM.
Infection with AV.GFP3ori was performed with 80% confluent
fibroblasts at an MOI of 1000 DNA particles/cell in 2% FBS DMEM for
24 hours.
[0186] S.sup.35 labeling of rAAV. The methionine residue in the
capsid protein of rAV.GFP3ori was labeled during the generation of
radioactive viral stocks according to a previously published
protocol with modifications (Mizukami et al., 1996). Briefly,
twenty 150 mm plates of subconfluent 293 cells were infected with
Ad.LacZ (5 pfu/cell) for 1 hour followed by calcium phosphate
transfection of pCisAV.GFP3ori (250 .mu.g) and pRepCap (750 .mu.g).
Cells were incubated for an additional 10 hours, at which time the
medium was changed to 2% FBS Methionine-free DMEM for 45 to 60
minutes. The medium was changed once again to labeling medium
containing 15 mCi of S.sup.35-methionine per 400 ml of 2% FBS
Methionine-free DMEM (final=1.49 MBq/ml), and cells were pulsed for
1.5 hours at 37.degree. C. Following labeling, L-methionine was
added back to a final concentration of 30 mg/L, and cells were
incubated for an additional 30 hours at 37.degree. C. Cell lysates
were prepared and virus was purified by isopycnic cesium chloride
ultracentrifugation as described above. Typical specific activities
of labeled virus preparations were 5.times.10.sup.-6 cpm/particle,
which is slightly higher than the 5.5.times.10.sup.-7 cpm/particle
specific activity reported by other investigators (Bartlet et al.,
1999).
[0187] Viral binding/entry assays and in situ localization of viral
particles. To assess the binding of rAAV to polarized bronchial
epithelia cells, S.sup.35-labeled AV.GFP3ori was applied to either
the apical or basal surface (MOI=50,000 particles/cell), followed
by incubation at 4.degree. C. for 60 minutes. Combined
binding/entry of rAAV into differentiated airway epithelia was
measured under the same conditions, except that the cultures were
incubated at 37.degree. C. for an additional 2-24 hours before they
were harvested. These combined viral binding/entry assays were
performed under identical infection conditions to those used for
functional studies of rAAV transduction with transgene expression
as an endpoint. After washing three times in PBS, cells were lysed
in situ by the addition of 5 ml of liquid scintillation cocktail at
room temperature for 5 minutes, and the radioactivity was
quantitated in a scintillation counter.
[0188] To analyze the subcellular localization of the rAAV
particles within polarized human bronchial epithelial cells,
infection was performed by applying S.sup.35 labeled virus
(MOI=50,000 particles/cell) to either the mucosal or serosal
surface. At 2 hours post-infection, transwells were washed with
medium three times and fixed in 4% paraformaldehyde overnight prior
to cryoprotection and embedding for frozen sectioning. 10 .mu.m
frozen sections were overlaid with photoemulsion and developed for
5 weeks according to a previously published protocol (Duan et al.,
1998).
[0189] Molecular analysis of rAAV viral genomes following infection
of polarized airway epithelial cultures. The molecular state of
bound and endocytosed virus was assayed at different times
following rAAV infection. To examine the amount of virus attached
to the cell surface, rAAV infection was performed at 4.degree. C.
for 1 hour. Following binding, the extent of viral internalization
was assessed by continuing incubations in the presence of virus at
37.degree. C. for 4-24 hours. Viral DNA was extracted according to
a modified Hirt protocol and Southern blots performed with Hybond
N+ nylon membrane (Amersham) (Duan et al., 1997). The 1.6 kb single
stranded viral DNA, the 2.7 kb double stranded circular
intermediate, and the 4.7 kb double stranded replication from viral
genome were detected with a transgene EGFP specific probe at
5.times.10.sup.6 cpm/ml. Blots were washed at a stringency of
0.2.times.SSC/0.1% SDS at 55.degree. C. for 20 minutes twice. In
studies aimed at evaluating viral internalization, virus attached
to the cell surface was removed by trypsinization with 1 ml of
buffer containing 0.5% trypsin, and 5.3 mM EDTA at 37.degree. C.
for 10 minutes (500 .mu.l buffer was added to the apical and
basolateral compartment of the Millicell inserts), followed by
washing with ice-cold PBS twice. Externally bound AAV virus was
determined by the intensity of the 1.6 kb viral genome band in Hirt
DNA extracted from cells infected at 4.degree. C. for 60 minutes.
The internalized virus was determined by the intensity of the 1.6
kb viral genome band in Hirt DNA extracted from trypsinized cells
after infection at 37.degree. C. for 4 and 24 hours. The dynamic
changes in the molecular structure of the internalized virus were
assayed at 2, 10, 30 and 50 days after virus was removed from
culture medium.
[0190] Detection of ubiquitinated AAV capsid proteins by
immunoprecipitation. To analyze the effect of the proteasome
inhibitor on AAV ubiquitination, human primary fibroblasts were
lysed at 6 hours post-viral infection in 1X RIPA buffer. Cell
lysates were then cleared with 30 .mu.l Protein A-Agarose. The
supernatant was incubated with 10 .mu.l of monoclonal anti-VP1, 2,
and 3 antibody (Clone B1, ARP) followed by the addition of 30 .mu.l
Protein A-Agarose. The pellets were washed 4 times with IX RIPA
buffer and resolved on a 10% SDS-PAGE. After transfer to a
nitrocellulose filter, blots were probed with a 1:1000 dilution of
anti-ubiquitin monoclonal antibody (clone P4D1, Santa Cruz,
catalogue #sc-8017), followed by 1:500 HRP-conjugated secondary
antibody (BMB). After the final washings, immunoreactivity was
visualized using the ECL system (Amersham).
[0191] In vivo studies in mice. Animal studies were performed in
accordance with the institutional guidelines of the University of
Iowa. To determine the effect of the proteasome inhibitor on AAV
mediated gene transfer in mouse lung, 6 week-old BALB/c mice were
lightly anesthetized using a methoxyflurane chamber. AV.LacZ
(5.times.10.sup.10 particles) was administered alone or with 400
.mu.M Z-LLL in a 10 .mu.l instillation by nasal aspiration as
described by Walters et al. (2000). To prevent unforeseen toxicity
of DMSO solvent, the proteasome inhibitor Z-LLL was dissolved in
ethanol as a 40 mM stock solution and was included in the viral
inoculum at 1% final concentration. Viral infection controls in the
absence of Z-LLL also contained a 1% final concentration of
ethanol. Since studies in both primary cultured human airway cells
and fibroblasts have demonstrated similar enhancement efficiency
between 40 .mu.M LLnL and 4 .mu.M Z-LLL, and also due to the poor
solubility of LLnL in ethanol (a low dose in DMSO had previously
been administered to the trachea), only Z-LLL was tested in this
particular mouse lung study. The animals were euthanized at 2, 10
and 150 days post infection and PBS (10 ml) was instilled into the
right ventricle, followed by removal of the lungs and heart as an
intact cassette. The trachea was intubated and instilled at 10 cm
of water pressure with the following solutions in order: PBS, 0.5%
glutaraldehyde, 1 mM MgCl.sub.2/PBS, and finally X-gal staining
reagent for an overnight incubation at room temperature. The X-gal
stained mouse lungs were then post fixed in 10% neutral buffered
formalin for 48 hours at room temperature and cryopreserved in
serial 10%, 20% and 30% sucrose/PBS solutions. Lungs (N=3 for each
condition) were embedded in OCT (optimal cutting temperature;
Baxter, Warrendale, Pa.) and 15 .mu.m serially sections were
analyzed for gene transfer by calculating the percentage of
positive cells in the airway epithelium. The diameter of the airway
was recorded for classification (>360 .mu.m, 260-350 .mu.m,
160-250 .mu.m, <150 .mu.m) of results following morphometric
analysis. Greater than 150 airway cross-sections were quantified
for each experimental condition.
Results
[0192] Molecular analysis of rAAV genomes in polarized airway
epithelia. Recent studies revealed a lack of AAV-2 receptor,
heparin sulfate proteoglycan, and co-receptors, FGFR-1 and
.alpha.V.beta.5 integrin, at the apical surface of differentiated
airway epithelia (Duan et al., 1998; Duan et al., 1999; Hughes et
al., 1993; Goldman et al., 1999). However, differences in the
binding of radioactive virus at the apical and basolateral
membranes were only 4-7 fold (basolateral>apical) (Duan et al.,
1998). These differences in binding are insufficient to explain the
200-fold variance observed in the polarity of infection
(basolateral>>apical) with rAAV-2 (Duan et al., 1998). These
findings suggested that viral binding and/or uptake were not the
sole limiting factors contributing to inefficient mucosal
transduction in airway epithelia. To this end, the molecular state
of rAAV DNA at 50 days following apical and basolateral infection
of air-liquid interface cultured human bronchial epithelia was
evaluated. At this time point, gene expression measured from an
EGFP reporter was >200-fold higher in basolaterally infected
cultures (data not shown) (Duan et al., 1998). Hirt DNA from the
cultures was evaluated by Southern blot hybridization with
.sup.32P-labeled EGFP probes. A significant amount of apically
applied rAAV was able to infect airway cells. However, only single
stranded viral genomes (ssDNA) were detected at this time point (50
days). This result clearly suggests that rAAV can be endocytosed
from the mucosal surface and that the endocytosed viral ssDNA was
stably sequestered in some unknown subcellular compartment. In
contrast, the majority of basolaterally applied rAAV was converted
into double stranded forms that migrated at 2.8 kb and >12 kb in
1% non-denaturing agarose gels. Consistent with previous reports
(Sanlioglu et al., 1999; Duan et al., 1999), subsequent restriction
enzyme mapping of Hirt DNA and Southern blots confirmed this 2.8 kb
band to be a supercoiled, circular episomal molecule (data not
shown). The identity of the >12 kb band, which is significantly
more intense following basolateral infection, is currently unknown
but may represent episomal circular concatamers of the AAV genome.
Taken together, these results suggest that inefficient molecular
conversion of AAV viral DNA to circular genomes represents a
significant obstacle for rAAV mediated gene transfer from the
apical surface of the airway. Furthermore, circularization, not
linear replication though self-priming, is the predominant pathway
for rAAV gene conversion in polarized airway epithelia.
[0193] Proteasome inhibitors dramatically enhance rAAV infection in
polarized airway epithelia. Given the fact that rAAV appears to
remain latent within some cellular compartment(s) following apical
infection in the airway, and that agents that alter the molecular
conversion of the viral genome might enhance rAAV transduction in
airway epithelia, several agents were tested in this regard,
including DNA damaging agents (Alexander et al., 1994), DNA
synthesis and topoisomerase inhibitors (Russell et al., 1995), and
cellular tyrosine kinases inhibitors (Qing et al., 1997; Man et
al., 1998). Application of camptothecin, etoposide, hydroxyurea,
and genistein resulted a 10 to 60 fold enhancement in rAAV
transduction from the basolateral surface. Interestingly, however,
none of these agents facilitated rAAV transduction from the apical
surface (data not shown). Since chemicals known to affect
intra-nuclear events involved in rAAV transduction in other cell
types (Sanlioglu et al., 1999) did not enhance rAAV apical
infection in the airway, other agents affecting endocytic
processing, such as the ubiquitin-proteosome pathway, were tested.
Proteasome systems are known to modulate the intracellular
processing of many foreign and endogenous molecules, including
viruses such as HIV (Schwartz et al., 1998). Several specific, cell
permeable, peptide aldehyde inhibitors of proteasome pathways have
recently been discovered (Rock et al., 1994; Fenteany et al.,
1995). These inhibitors bind to the active sites of proteolytic
enzymes within the proteasome core and reversibly block their
function (Rubin et al., 1995). To test whether proteasomes
represent an intracellular compartment that sequesters rAAV
following infection, the tripeptidyl aldehyde proteasome inhibitor
(a cysteine protease inhibitor)
N-acetyl-L-leucinyl-L-leucinal-L-norleucinal (LLnL, also called
Calpain inhibitor I) was applied to polarized cultures of human
bronchial epithelial cells at the time of rAAV infection.
Surprisingly, a greater than 200 fold augmentation in transgene
expression was obtained at 2 days post infection when 40 .mu.M LLnL
was applied to the serosal surface along with rAAV. A similar
result was achieved when another ubiquitin-proteasome pathway
inhibitor, N-carbobenzoxyl-L-leucinyl-L-leucinyl-L-leucinal (Z-LLL,
also called MG132) (Jensen et al., 1995), was tested (data not
shown). However, the most important finding was that these
proteasome inhibitors also substantially increased rAAV
transduction from the mucosal surface (see below). When compared
with other agents, proteasome inhibitors were found to be the most
potent enhancers of rAAV transduction in airway epithelium.
[0194] Proteasome inhibitors augment rAAV transduction in airway
epithelia in a polarized fashion. Although proteosome inhibitors
appear to significantly increase the efficacy of rAAV transduction
from the serosal surface, the route most germane to clinical
application of gene delivery in the airway is the mucosal surface.
To test the effect of proteasome inhibitors on rAAV transduction
from apical membrane, a side-by-side kinetic comparison of rAAV
transduction from both mucosal and serosal surfaces of airway
epithelia following treatment with LLnL was performed.
Co-administration of LLnL and rAAV to the mucosal surface resulted
a sustained augmentation in AAV transduction, which peaked at 22
days post-infection. In contrast to mucosal infection, rAAV
infection from the serosal surface in the presence of LLnL resulted
only in a transient peak in gene expression at 72 hours
post-infection, which returned to the levels equivalent to that of
the untreated samples by 22 days. These results suggested that the
proteasome inhibitor LLnL produces different augmentation profiles
when AAV virus is applied to either the apical or the basolateral
membranes. To exclude potential effects caused by polarized uptake
of LLnL by airway epithelia, different combinations of rAAV and
LLnL administration from both apical and basolateral surfaces were
tested. Similar augmentation patterns for AAV transduction were
achieved, regardless of whether LLnL was applied to the same or
opposite surface as rAAV during infections (data not shown).
[0195] To determine whether LLnL administration augmented rAAV
transduction of particular airway cell types, a rAAV vector
encoding the alkaline phosphatase gene (Alkphos) was utilized.
Transduced cell types were evaluated by standard histochemical
staining for Alkphos to address this question. In the absence of
LLnL, rAAV preferentially transduced basal cells at 3 days
following serosal application of virus. Consistent with previous
findings utilizing AV.GFP3ori virus, co-administration of LLnL
resulted in a dramatic increase in AV.Alkphos transduction.
Interestingly, ciliated cell transduction was most significantly
increased by treatment with LLnL at the time of rAAV infection. In
contrast, basal cells were the least responsive to LLnL treatment.
These findings indicated that the mechanisms of LLnL action may
have some cell specific components, which differs in polarized
(i.e., ciliated) and non-polarized (i.e., basal) cell types.
[0196] Optimization of LLnL enhanced rAAV transduction. With the
aim of further improving the enhancement in rAAV transduction
achieved in the presence of LLnL, several detailed kinetic studies
were performed which altered the timing and number of LLnL
administrations following rAAV infection. Several important
conclusions arose from these studies. First, following basolateral
infection, administration of LLnL once every three days increased
length of peak transgene expression, despite the fact that by the
end of 30 days levels were similar to that of cultures treated once
at the time of infection. Second, continual administration of LLnL
was toxic to cells and ablated transgene expression by 10 days.
Third, re-infection of cultures with rAAV in the presence of LLnL
at 7, 10 and 15 days resulted in a similar pattern of augmentation
and, as expected, elevated the final level of transgene expression
observed at 30 days (only data from the second infection at 15 days
are shown). Most notably however, all the cultures infected from
the basolateral side produced similar long-term transgene
expression levels within 2 to 3 fold of each other, regardless of
whether LLnL was administered.
[0197] Despite the fact that LLnL administration at the time of the
viral infection augmented rAAV transduction from both the apical
and basolateral surfaces, the kinetics of this induction were
significantly different. Enhancement following basolateral
infection was transient, while enhancement following apical
infection was long-term. Furthermore, although induction with LLnL
from the apical membrane was long-lasting, by 30 days the maximal
level of transgene expression was only one eighth of that resulting
from basal infection. The application of hypotonic EGTA solution
has been shown to increase AAV transduction from the apical surface
by 7 to 10 fold (Duan et al., 1998; Walters et al., 2000).
Therefore the combined administration of EGTA and LLnL could
provide yet a further increase in rAAV transduction from the apical
surface. Interestingly, treatment of airway cultures with EGTA
prior to infection with rAAV in the presence of LLnL gave a
transient peak in transduction within the first three days, and a
significantly increased (200-fold), prolonged level of transgene
expression out to 30 days. This prolonged level of transgene
expression, while comparable to rAAV infection from the basal
surface, was much above the level observed in apically infected
epithelia treated with EGTA alone. In summary, these results
demonstrate that EGTA and LLnL have synergistic effects on rAAV
transduction, allowing for transduction from the apical surface at
levels normally only seen following basolateral infection.
[0198] Viral binding and internalization are not affected by LLNL
treatment. The action of LLnL has been typically attributed to it
selective and reversible inhibition of the proteasome system.
However, it was important to rule out any possible effect on viral
binding and/or endocytosis. As has been found for type I herpes
simplex virus (Everett et al., 1998), LLnL treatment had no
significant effect on 4.degree. C. rAAV binding. Similarly, the
uptake of S.sup.35 labeled rAAV for a 2 hour infection period at
37.degree. C. was not altered by LLnL treatment. Given these
results, LLnL acts at points distal to virus binding and entry.
Interestingly, at 24 hours post-infection a very significant
decrease in the amount of intracellular radioactivity was observed
in epithelia treated with LLnL, regardless of which surface was
infected. Given the concordant increase in transgene expression at
this time point, LLnL may be accelerating processing and routing of
the virus to the nucleus, wherein uncoating and clearance of
S.sup.35 labeled capsid proteins occur. By this mechanism, S.sup.35
isotope would be diluted into the culture medium and could explain
the decrease in cell-associated counts.
[0199] LLnL enhances endosomal processing and nuclear trafficking
of rAAV. To test the hypothesis that LLnL increases trafficking of
rAAV to the nucleus, in situ localization of the S.sup.35-labeled
rAAV particles following infection from the apical and basolateral
surfaces was performed in the presence and absence of LLnL. Since
loss of intact radiolabeled capsid proteins occurred at 24 hours
post-infection, a 2 hour time point was chosen for this analysis.
Using photoemulsion overlay, the subcellular distribution of
S.sup.35-labeled rAAV particles was evaluated by blinded
morphometric analysis. The majority of viral particles localized to
the cytoplasm in the absence of LLnL. This was the case regardless
of whether infection was performed from the apical or basolateral
surface. In contrast, LLnL treatment substantially changed the
intracellular distribution of radiolabeled rAAV particles,
resulting in a significant shift to nuclear associated grains.
These results substantiated the findings from whole cell counts at
24 hours post-infection, which suggested that LLnL increases viral
uncoating and the subsequent loss of S.sup.35 isotope into the
media.
[0200] LLnL augment rAAV transduction within a specific time frame
after infection. Evidence thus far has suggested that LLnL may act
to increase intracellular routing of rAAV to the nucleus.
Additionally, LLnL action is independent of the epithelial surface
to which it is administered (i.e., serosal application of LLnL
augments mucosal infection and vice versa). This indicates that
LLnL need not be endocytosed with AAV particles to enhance
transduction. Thus, LLnL may act at a specific time following rAAV
endocytosis but during endosomal processing. To provide functional
support for this hypothesis, a kinetic analysis of LLnL action at
various times after infection from the basolateral surface was
performed. In these experiments, LLnL was added to the culture
medium either at the time of AAV infection or at various time
points after infection. Viral-mediated transgene expression was
quantified at 24 hour intervals following infection. Augmentation
was achieved regardless of whether LLnL was administrated at 0, 24,
48, and 72 hours after viral infection. However, addition of LLnL
at 24 or 48 hours gave the strongest level of augmentation. The
ability of LLnL to reduce AAV expression appeared to decline by 72
hour post-infection and was completely lost by 15 days after the
initial AAV infection (data not shown). Taken together, it appears
that after rAAV enters the cell, it may be targeted to an
intracellular compartment that is sensitive to proteasome
inhibitor-facilitated liberation. In addition, the loss of an LLnL
augmentation effect at 15 days post-infection suggests that
enhanced transcription, translation, and/or stability of the
transgene products do not likely contribute to the mechanism
responsible for this observation.
[0201] Combined treatment of LLnL and EGTA prevents degradation of
internalized rAAV. To further clarify the molecular mechanism(s)
responsible for augmentation of rAAV transduction by LLnL, rAAV
genomes in infected cells were analyzed by Southern blotting Hirt
DNA. Consistent with studies using S.sup.35 labeled virus, rAAV
binding to either surface of polarized airway epithelia was not
affected by LLnL treatment. Southern blotting also demonstrated 2
to 7 fold higher viral binding from the basal surface, which varied
among different tissue samples (data not shown). The extent of
virus internalization was compared after stripping surface bound
virus with trypsin. Confirming previous results, a significant
amount of rAAV was endocytosed from the apical surface during the
infection period, although viral uptake was more active from
basolateral surface. LLnL alone also did not substantially prevent
enzymatic degradation of the internalized AAV viral DNA, indicating
that enhanced viral trafficking into the nucleus might be more
important in the observed augmentation by LLnL. However, treatment
with both hypotonic EGTA and LLnL substantially increased the
amount of virus internalized from apical surface. Since hypotonic
EGTA treatment alone only slightly increased persistence of AAV DNA
or AAV-mediated gene expression (Duan et al., 1998; Walters et al.,
2000) following apical infection, the predominant mechanism
responsible for the combined effects of EGTA and LLnL might be due
to reduced degradation of the internalized virus and an increased
rate of endocytosis. These synergistic effects of EGTA and LLnL
augment rAAV transduction from the apical membrane more than
200-fold. Additionally, the conversion of single stranded viral
genomes to linear replication or circular forms has been associated
with enhanced AAV transduction by adenoviral early gene products or
UV irradiation, respectively (Fisher et al., 1996; Sanlioglu et
al., 1999; Duan et al., 1999). Southern blots of Hirt DNA from
cultures co-infected with Ad.d1802 and rAAV showed LLnL enhanced
AAV transduction was clearly not mediated through the formation of
linear replication intermediates (4.7 kb band) as seen in the
presence of adenoviral E4orf6 protein produced by Ad.d1802
co-infection.
[0202] Ubiquitination of viral capsid proteins following rAAV
infection in the airway alters the efficiency of transduction.
Proteasome-dependent degradation of ubiquitinated molecules
represents a major pathway for disposal of both endogenous and
foreign proteins (Schwartz et al., 1999). Several distinct steps in
the regulation of this pathway have been identified, including:
activation of ubiquitin by its activating enzyme (E1), transfer of
the activated ubiquitin to the ubiquitin carrier protein (E2), and
subsequent delivery of the activated ubiquitin to the protein
substance by ubiquitin ligase (E3). Ultimately, ubiquitinated
proteins are degraded by the 26S proteasome through an
ATP-dependent process. To test whether enhancement of rAAV
transduction by proteasome inhibitors involves liberation of
ubiquitinated virus from an endosomal compartment, the extent of
ubiquitin side chains on AAV capsid proteins following infection
was examined as well as whether treatment with proteasome
inhibitors altered the extent of ubiquitination. AAV capsid
proteins were immunoprecipitated using anti-VP 1, 2, 3 antibody
from rAAV infected human polarized airway cells and confluent human
fibroblasts at 6 hours post-viral infection. Subsequent Western
analysis with anti-ubiquitin specific antibodies demonstrated a
significant increase in the cellular level of ubiquitinated AAV
capsid in fibroblasts following proteasome treatment.
Ubiquitination significantly increased the molecular weight of
capsid proteins (63 kd, 73 kd, and 87 kd) to 220-250 kd and is
consistent with the size change following ubiquitination for other
molecules (Bregman et al., 1996). Unfortunately, the limited amount
of virus retrievable from air-liquid interface cultured human
airway cells precluded the ability to detect ubiquitinated capsid
in this system (data not shown). Nonetheless, confluent primary
fibroblasts also demonstrated augmentation (10-fold) of transgene
expression following treatment with proteasome inhibitors. Thus,
proteosome inhibitors increase rAAV transduction by decreasing the
targeting and/or degradation of ubiquitinated AAV in the
proteosome. The net result of such proteasome inhibition would be
expected to increase the abundance of ubiquitinated viral
capsid.
[0203] Because a technical limitation in polarized airway model
prevented direct detection of ubiquitinated viral capsid, it was
determined whether modulation of other steps in the ubiquitin
proteasome pathway could also increase rAAV transduction similarly
to that seen with proteasome inhibitors LLnL and Z-LLL. Several
dipeptides, such as H-Leu-Ala-OH and H-His-Ala-OH, are known to
inhibit ubiquitin ligase E3 (Obin et al., 1999). Application of
these ubiquitin ligase inhibitors indeed enhanced rAAV transduction
from the basolateral surface of human airway cells. Taken together,
data in both fibroblasts and polarized airway epithelia suggest
that AAV capsid is ubiquitinated following endocytosis, and that
this process is a barrier to rAAV transduction. The most plausible
mechanism responsible for the augmentation of rAAV transduction by
tripeptide proteasome inhibitors involves the prevention of
ubiquitinated virus degradation and/or targeting to the
proteasome.
[0204] Long-term enhancement of rAAV transduction by proteasome
inhibitor in vivo. To evaluate the potential utility of proteasome
inhibitors for in vivo gene therapy, both the toxicity and efficacy
of these agents for in vivo rAAV mediated gene transfer in the
mouse lung was tested. To assess the toxicity of these proteasome
inhibitors in mice, 10, 100, and 1000 fold higher effective doses
of LLnL or Z-LLL were administered than used to induce gene
transfer in polarized airway cells, using both intra-tracheal and
systemic (IV) delivery. No toxicity was indicated by histologic
evaluation of the lung and liver or was evidenced by the death of
animals. To investigate whether these proteasome inhibitors could
improve rAAV transduction in vivo, AV.LacZ (5.times.10.sup.10
particles) was delivered either alone or in the presence of 400
.mu.M Z-LLL by intranasal administration. Mouse lungs were
harvested at 3, 10 and 150 days post-infection to evaluate short
and long term effects. Proteasome inhibitor treatment from basal
surface, or in conjunction with EGTA from apical surface, resulted
in pronounced, immediate enhancement on rAAV transduction, however,
X-gal staining of the lung tissues at 3 and 10 days post infection
demonstrated no detectable transgene expression in either
proteasome inhibitor treated or untreated groups. In contrast,
significant transduction was achieved at 150 days in Z-LLL treated
samples. Targeted transgene expression was predominantly confined
to the conducting airways, rather than in the parenchyma. Alveolar
cells were rarely transduced. Although on average only about 5.88%
of airway cells were transduced by AV.LacZ, and LacZ positive cells
were observed throughout the entire conducting airway, a
characteristic transduction profile was evident. The transduction
efficiency in larger bronchioles (>350 mm) reached a mean of
10.36.+-.1.63% of the airway epithelium, while 1.37.+-.0.41% of
airways cells in the smaller bronchioles (<150 mm) expressed the
.beta.-galactosidase transgene. The range of transgene expression
in distal and proximal airways was 0 to 4% and 5 to 18%,
respectively. This transduction profile demonstrating a higher and
more consistent transduction in larger airways likely reflects a
more uneven delivery of virus to regions of the lung encompassing
the smaller airways. Examination of cryo-sections from lungs
infected by AV.LacZ alone revealed only 2 lacZ positive cells in a
total of 315 airway sections (n=3 animals).
Discussion
[0205] Inefficient gene transfer from the apical surface of the
airway has been a major obstacle in numerous gene therapy
approaches for cystic fibrosis utilizing recombinant adenovirus
(Walters et al., 1999; Pickles et al., 1998), adeno-associated
virus (Duan et al., 1998), retrovirus (Wang et al., 1998), and
non-viral liposome vectors (Chu et al., 1999). It has been
generally thought that inefficient viral mediated gene delivery
through the apical membrane of airway epithelia is predominantly
due to the lack of receptors or co-receptors on this surface.
[0206] Molecular analysis of rAAV infection in polarized airway
epithelia has revealed several unique findings. First, there is
conclusive evidence that the previously reported lack of known
AAV-2 receptor and co-receptors (Duan et al., 1999) at the apical
membrane of airway epithelia does not abrogate AAV infection.
Although transduction (as determined by transgene expression) from
the basolateral surface is 200-fold more efficient than from the
apical membrane, quantitative and semi-quantitative analyses of
viral endocytosis with either S.sup.35-labeled virus or Southern
blotting have demonstrated that viral uptake from the apical
surface is only 2-7 fold less efficient than from the basolateral
membrane. Therefore, it is reasonable to assume that previously
unidentified alternative receptor/co-receptors and/or
receptor-independent mechanism(s) might be involved in AAV uptake
from the mucosal surface of the airway.
[0207] Polarity is widely recognized to significantly influence
endosomal processing of many proteins, and distinct sorting
mechanisms have been described for the apical and basolateral
compartments (Odorizzi et al., 1996; Rodriguez-Boulan et al.,
1993). The lack of a direct correlation between the efficiency of
viral uptake and transgene expression following basolateral and
apical infection suggest that additional post-endocytic barriers
exist for rAAV mediated gene transfer. Differences in the extent of
AAV nuclear trafficking following basolateral versus apical routes
of infection suggest that basal and apical cellular compartments
possess distinct biologic properties that may influence the
polarity of AAV transduction. Endosomal processing barriers to rAAV
transduction may not be limited to polarized epithelial cells. In
support of this notion, impaired intracellular trafficking of viral
particles to the nucleus has been observed in NIH 3T3 cells. In
addition, rAAV can remain in an inactive state for as long as 7
days in confluent primary fibroblast cells until rescued by UV
irradiation to a functionally active state. Thus, post-endocytic
barriers to infection exist in multiple cell types.
[0208] In the airway, the major rate-limiting steps in rAAV
transduction from the mucosal surface appear to involve inefficient
endosomal processing of the internalized virus. Regulated
intracellular proteolysis through proteasomes plays a critical role
in many physiological and pathological conditions (Schwartz et al.,
1999; Kato, 1999). Recent identifications of many specific
proteasome inhibitors has set the foundation for pharmacologic
intervention in this cellular enzymatic system as a novel
therapeutic approach. For example, several cell permeable synthetic
tripeptide aldehydes (such as LLnL and Z-LLL used in this study)
have been demonstrated to be promising cancer therapy agents or
anti-inflammatory drugs (Goldberg et al., 1995; Kloetzel, 1998;
Wojcik, 1999). Additionally, the proteasome has been suggested to
have antiviral functions in HIV infection (Schwartz et al., 1998),
implying that the inhibition of proteosome function could be
beneficial in promoting transduction with recombinant viruses.
Based on the molecular evidence that apical infection of rAAV in
the airway is significantly limited by post-entry events,
ubiquitin/proteasome pathways appear to be instrumental in this
blockage. The proteasome is commonly know as a compartment for
clearance of endogenous and foreign proteins. However, recent
studies also suggested that the proteasome system is involved in
regulating endocytosis (Bonifacino et al., 1998; Strous et al.,
1999). From the standpoint of gene delivery, proteasome inhibitors
have been shown to protect transfected plasmid DNA from degradation
(Coonrod et al., 1997). The results described herein clearly
demonstrate that rAAV mediated gene transfer to the airway
epithelia is also significantly enhanced by proteasome inhibitors.
Furthermore, this enhancement is correlated with proteasome
inhibitor stimulated viral trafficking to the nucleus. Although
proteasome inhibitors increased long-term levels of AAV
transduction form the apical surface, their effect on basolateral
infection appeared predominantly to alter the rate, rather than the
long-term levels, of transduction. These differences highlight
fundamentally distinct pathways involved in rAAV transduction from
apical and basolateral surfaces.
[0209] Several findings also support the notion that ubiquitination
of virus following endocytosis may be a critical mechanism for
sorting incoming AAV. First, treatment of airway epithelia with
proteasome inhibitors know to block ubiquitin-dependent degradation
of proteins enhances rAAV gene transfer. Second, inhibition of
ubiquitin E3 ligase activity in airway epithelia also enhances
transduction. Lastly, rAAV capsid proteins are ubiquitinated
following infection in confluent human fibroblasts, and that the
extent of this ubiquitination is increased by inhibition of
ubiquitin-proteasome degradative pathways.
[0210] From an applied standpoint, one of the most important
findings in this study is the persistent high level of rAAV
transduction induced by proteasome inhibitor in mouse lung.
Co-administration of Z-LLL with rAAV increased transgene expression
from undetectable levels to 10.36+/-1.63% of proximal bronchial
epithelial cells at 150 days post-infection. This level of gene
expression is thought to be sufficient for therapeutic correction
of CFTR deficiency (Crystal, 1999). The feasibility of this
strategy for clinical application is further supported by the lack
of a detectable local or systemic toxicity following proteasome
inhibitor administration to mice. Furthermore, preliminary studies
in several other organs, e.g., heart skeletal muscle and liver,
have suggested that ubiquitination of rAAV2 may occur in an
organ-specific fashion. The application of proteasome inhibitors in
skeletal and cardiac muscle had no effect on either short-term or
long-term rAAV mediated gene transfer. However, application of
Z-LLL in the liver led to a 7-fold increase in rAAV transduction at
1 month post-infection. These findings suggest that tripeptide
proteasome inhibitors could be used to increase gene transfer in
organs other than the lung, depending on the cell biology of virus
processing.
[0211] In conclusion, a significant barrier to apical infection in
the airway with rAAV-2 lies at the level of endosomal processing
and ubiquitination. Modulation of the ubiquitin-proteasome system
has revealed innovative strategies to enhance rAAV transduction
from the mucosal surface of the airway for gene therapy of cystic
fibrosis.
EXAMPLE 2
Expression of the LacZ Gene in Lung
Airway Epithelium and Liver In Vivo
[0212] The in vivo activity of rAAV in the presence or absence of
an agent of the invention in the lung or liver may be tested using
the LacZ gene. A rAAV vector containing the LacZ gene, recombinant
AV.LacZ (about 5.times.10.sup.10 particles), was administered to
mouse lung either as virus alone in PBS or virus in combination
with 40 .mu.M LLnL in PBS. Virus was directly instilled into
C57Balb/c mice trachea with a 30 G needle in a total volume of 30
.mu.l. To insure the spread of the virus in mouse lung, 50 .mu.l
air was pumped into lung through the same syringe immediately after
virus was administrated. Ninety days after infection, lungs were
harvested intact and fixed in 4% paraformaldehyde followed by
cryosection. AAV-mediated transgene expression was evaluated by 10
.mu.m tissue sections staining for LacZ.
[0213] Recombinant AV.LacZ (about 5.times.10.sup.10 particles) was
also administered to mouse liver either as virus alone in PBS,
virus in combination with 40 .mu.M Z-LLL in PBS, or virus in
combination with 20 .mu.M LLnL in PBS. Virus was directly instilled
into portal vein of the C57B6 mice. AAV-mediated LacZ transgene
expression was evaluated by histology staining at 2 and 4 weeks
post infection in frozen tissue sections.
EXAMPLE 3
Methods to Determine Additional Agents Useful to Enhance rAAV
Transduction
[0214] A. To screen for agents that enhance rAAV transduction, any
number of cells can be used. A range of concentrations of the agent
to be tested can be determined based on, for instance desirable
profiles of the agent, desirable toxicity profiles of the agent
and/or concentration of the agents employed in vivo. The usefulness
of the cell type chosen for the screen can be confirmed by testing
compounds, e.g., proteosome inhibitors described in Example 1 such
as LLnL and ZLL which are known to increase rAAV transduction. For
example, a AAV2 FLAG-Luc vector was employed to transduce HeLa,
ferret fibroblasts, IB3 and Huh (liver) cells in the presence or
absence of the proteosome inhibitor MG132. MG132 was confirmed to
enhance AAV transduction in all cell types tested: HeLa cell
transduction was enhanced about 500-fold at 80 .mu.M, and 200-fold
at 40 .mu.M, MG132; ferret fibroblast cell transduction was
enhanced about 200-fold at 20 .mu.M, and 17-fold at 4 .mu.M, MG132;
IB3-1 cell transduction was enhanced about 30 to 70-fold at 20 to
80 .mu.M MG132; and Huh-7 cell transduction was enhanced about
15-fold at 20 to 80 .mu.M MG132. There was no difference in rAAV
transduction efficiency in HeLa cells when either DMSO or ETOH was
used as a vehicle for MG132. B. HeLa cells were selected to screen
for additional agents that enhance rAAV transduction, although any
cell strain or line; or primary cells, may be employed. Agents were
selected from various classes, such as anti-inflammatories (e.g.,
dexamethasone and cyclosporin A), NSAIDs (e.g., ibuprofen),
.beta.-adrenergics (e.g., albuterol), antibiotics (e.g.,
ciprofloxacin, colison, gentamycin, tobramycin, and epoxomycin),
lipid lowering agents (e.g., lovastatin, simvastatin and
eicosapentaenoic acid), food additives (e.g., tannic acid), viral
protease inhibitors (e.g., Norvir, Kaletra, and Viracept),
chemotherapeutics (e.g., aclacinomycin A, doxorubicin, doxil,
camptothecin, taxol and cisplatin) and protease inhibitors (e.g.,
chymostatin, bestatin and chloroquine). The range of concentrations
of the agents to be tested were selected based on solubility
profiles, toxicity profiles and/or concentrations previously
employed in vivo.
[0215] HeLa cells were infected for 2 hours with an MOI of 100 rAAV
in the presence of agents, e.g., ritonavir (Norvir) (1, 10 and 100
.mu.M), cyclosporin A (2.5, and 250 .mu.g/ml), epoxomicin (1, 10
and 50 .mu.M), alcacinomycin A (5, 50 or 500 .mu.M), chymostatin
(1, 10 and 100 .mu.M), bestatin (1, 10 and 100 .mu.M), doxorubicin
(adriamycin) (0.1, 1 and 10 .mu.M), camptothecin (camptosar) (1, 10
and 100 .mu.M), eicosapentanoic acid (1, 10 and 100 .mu.M), tannic
acid (2, 20, 200 and 2000 .mu.M), simvastatin, prodrug (2, 20 and
200 .mu.M), cisplatin (0.2, 2 and 20 .mu.g/mL), and chloroquine (4,
40 and 400 .mu.M). Forty-eight hours after infection, cells were
harvested for analysis. rAAV transduction was measured by removing
the media from the cell cultures, adding 100 .mu.L reporter lysis
buffer (RLB) and freezing. The supernatant was thawed and
transferred to microfuge tubes, freeze thawed an additional 2
times, clarified by centrifugation for 10 minutes and then analyzed
for reporter gene expression on the luminometer. Protein was
determined by Bradford analysis and results were expressed as
relative light units per mg protein (RLU/mg). Data is presented in
FIGS. 1A-E.
[0216] Doxorubicin, epoxomicin, and camptothecin all showed a
dose-dependent increase in transduction at the dose ranges tested.
At the doses tested doxorubicin and epoxomicin increased
transduction efficiency up to 169-fold and 120-fold, respectively,
camptothecin increased transduction efficiency by 15-fold, tannic
acid increased transduction efficiency by 17-fold, cisplatin
increased transduction efficiency by 16-fold, and simvastatin
increased transduction efficiency by 4-fold.
[0217] It should be noted with respect to simvstatin and the
lovastatin, that these drugs are formulated as prodrugs and
conversion to the activated open ring forms was not confirmed which
may have contribute to the negative results. Similarly, the
liposomal formulation of doxorubicin, doxil could not be confirmed
to be bioavailable to cell culture cells. Thus, agents which
initially screened as statistically negative may be reflective of
formulations that are not readily bioavailable to cell culture
cells or may be reflective of the limited dose range or exposure
time.
[0218] Epoxomicin, a naturally occurring antibiotic isolated from
Actinomycetes known to inhibit NF-KB-mediated signaling in vivo and
in vitro, inhibits proteosomes by inhibiting a proteosome-specific
chymotrypsin-like protease. Doxorubicin, an anti-tumor antibiotic
which inhibits topoisomerase II and inhibits nucleic acid
synthesis, is translocated by a 20S proteosome from the cytoplasm
to the nucleus. Camptothecin, a reversible DNA topoisomerase
inhibitor, down regulates topoisomerase via an ubiquitin/26S
proteosome pathway. Simvastatin is an agent that modulates
proteosome activity, tannic acid inhibits chymotrypsin-like
activity and is a cancer chemopreventative, and cisplatin is a
chemotherapeutic which crosslinks DNA.
C. To determine whether combinations of agents that enhance rAAV
transduction efficiency have synergistic or additive effects when
used in combination, cells were contacted with the proteosome
modulator, doxorubicin, and the proteosome inhibitor Z-LLL or LLnL.
Different AAV vectors were tested, including splicing vectors and
pseudotyped rAAV. Viral stocks utilized were as follows: Av2RSVluc,
5.times.10.sup.8 particle/.mu.l; Av2RSVlucCap5 (also referred to as
Av2/5 CMVLuc), 2.times.10.sup.9 particle/.mu.l; Av2CMVluc,
1.3.times.10.sup.9 particle/.mu.l; and Av2CMVlucCap5,
1.1.times.10.sup.9 particle/.mu.l. Combinations of agents were
compared to the agents used alone to determine the efficiency of
transduction. LLnL was used at 40, 200 or 400 .mu.M, Z-LLL at 4
.mu.M and doxorubicin at 0.5 or 1 .mu.M when employed alone. When a
combination of LLnL and doxorubicin was used, LLnL was used at 4,
10, 20, 40, 200 or 400 .mu.M and doxorubicin at 1 or 5 .mu.M. The
apical surface of polarized airway epithelia, HeLa cells or ferret
fibroblast was contacted with the agents and rAAV (5.times.10.sup.9
particles per well).
[0219] The results showed that LLnL enhances transduction in HeLa,
ferret fibroblast and polarized epithelial cells at 40 .mu.M and
A549 cells at 200 to 400 .mu.M. Doxorubicin enhanced transduction
in HeLa and ferret fibroblast cells at 1 .mu.M and A549 or
polarized airway cells at 5 .mu.M, and enhanced transduction about
100 fold when ferret fibroblasts were infected with lacZ splicing
vectors. Doxorubicin also enhanced AAV2 and AAV5 transduction to a
greater extent than LLnL. Synergistic effects were noted when
doxorubicin and LLnL were co-administered.
[0220] In the absence of agent administration, transduction from
the apical surface of polarized epithelial cells was greater with
AAV vectors with AAV5 capsid than AAV vectors with AAV2 capsid. In
the presence of doxorubicin, a 200 to 600-fold induction was
observed for AAV2 and AAV5 apical infection of polarized cells.
Thus, agents of the invention can enhance rAAV transduction,
including in serotype, pseudotype and multiple vector
strategies.
D. Endotracheal administration of 10.sup.11 AV2FLAG-luc rAAV
particles to male Balb/c mice in conjunction with intravenous
administration of Doxil (dosed in a range of 2, 10, or 20 mg/kg), a
liposomal preparation of doxorubicin, to mice enhanced AV2FLAG-luc
transduction by 2 logs by day 7 at the 20 mk/kg dose of doxil.
Specifically, at 20 mg/kg doxil, transduction was enhanced on the
average of 67-fold by day 7 and 4-fold by day 30 (FIG. 2). It is
worth noting that doxil previously tested negative in cell line
screening while the free compound doxorubicin tested positive in
cell line screening (FIGS. 1A-E). Liposomal formulations have
desirable properties for in vivo use including their increased
stability or circulation half life making them more bioavailable in
vivo. Those same characteristics make liposomal formulations less
desirable for in vitro screening as described above. Thus, one
skilled in the art can design formulation strategies for agents of
the invention to tailor them to the desired application. In
addition to formulation design, one skilled in the art can tailor
routes of delivery in order to maximize rAAV transduction
efficiencies.
[0221] In additional experiments, a pseudotyped rAAV vector
encoding FVIII was tested in male Rag-1 mice. Rag-1 mice were used
because as described in the art, normal mice produce inhibitors of
human FVIII that can obscure protein detection in the serum. Rag-1
mice are known to be deficient in the pathways necessary to produce
these inhibitors and thus will either produce no inhibitors, lower
levels of inhibitors or have extended time periods for development
of inhibitors. The rAAV vector was constructed containing serotype
5 capsid proteins and 5'-3' ITRs of AAV-2 flanking a heterologous
transgene comprised of the minimal liver specific element HNF3/EBP
and a human B-domain deleted FVIII gene (a second construct was
identical except it contained a B-domain deleted canine FVIII
gene). Animals were administered 10.sup.12 rAAV vector particles
intravenously via the lateral teil vein concurrently with 20 mg/kg
of doxil at day 0. Circulating, bioavailable FVIII activity was
measured from the serum at days 31, 53 and 90 by techniques known
in the art including ELISA and Coatest. Data presented in FIG. 3
demonstrate that animals not treated with doxil had barely
detectable levels of FVIII in the range of 0.99 ng/ml for days 31
and 53 which decreased to 0.13 ng/ml by day 90. In contrast,
animals dosed with 20 mg/kg of doxil had over 40 times the levels
of FVIII protein. Interestingly, the decline in FVIII protein seen
in animals not treated with doxil at day 90 (0.13 ng/ml) was not
evident in animals treated with doxil (40.16 ng/ml) indicating that
doxil not only enhanced rAAV transduction as evident at the shorter
time period, but the agent of the invention also prolonged
expression. In order to demonstrate that doxil was affecting rAAV
transduction and not merely affecting the FVIII protein translation
or stability, RS-PCR was performed on liver tissue at the day 53
time point. The data presented for individual animals in Table 1
demonstrates that the increase in FVIII protein noted in animals
treated with doxil correlates with the levels of mRNA detected.
[0222] The increase in vivo rAAV transduction produced by doxil was
further confirmed utilizing the same vectors and protocol described
above in male FVIII knockout mice tolerized to the human FVIII
protein utilizing a cytoxan mediated tolerization strategy as
described in the art. Animals were treated with weekly injection of
50 mg/kg cytoxan beginning at the time of rAAV vector delivery.
Data presented in Table 2 confirmed the previously described
results when tested by ELISA or Coatest at days 14 and 25, namely
animals dosed with doxil demonstrated at least a ten-fold
enhancement of rAAV transduction.
TABLE-US-00001 TABLE 1 Animal Molecules FVIII FVIII Protein Number
Treatment mRNA/cell (ng/mL) #26 AAV2/5 HNF3/EBP 2.15 0.68 #27 0.91
<0.63 #28 1.98 0.97 #29 2.06 1.45 #30 2.45 0.77 #31 2.29
<0.63 #59 AAV2/5 HNF3/EBP 65.47 31.85 #60 FVIII + Doxil 41.4
37.75 #61 99.43 51.9 #62 49.44 38.65 #63 43.9 40.55 #64 57.54 31.55
#26 2.15 0.68 #27 0.91 <0.63 #28 1.98 0.97 #29 2.06 1.45 #30
2.45 0.77 #31 2.29 <0.63 #59 65.47 31.85 #60 41.4 37.75 #61
99.43 51.9 #62 49.44 38.65 #63 43.9 40.55 #64 57.54 31.55
TABLE-US-00002 TABLE 2 In Vivo Enhancement of FVIII rAAV
Transduction Day 14 Results Animal # and Final Result Sample
(DF*ng/mL) Coatest (mU/mL) Group 1 Vehicle 801 < 0.63 0 804 <
0.63 0 805 < 0.63 0 847 < 0.63 0 Group 2 AAV2/5-HFN3/ 816
< 0.63 0 EBP-FVIII 817 < 0.63 0 818 0.92 0 819 < 0.63 0
820 < .63 0 834 0.9 0 Group 2 AAV2/5-HFN3/ 870 60.45 171
EBP-FVIII +Doxil 871 26.29 0 872 12.395 14 873 44.3 30 874 12.135
122 875 31.04 94 2.X.10, Day 25 FVIII ELISA Sample Animal # and
Final Result Group 1 Vehicle 806 < 0.63 0 807 < 0.63 0 808
< 0.63 0 849 < 0.63 0 Group 2 AAV2/5-HFN3/ 821 < 0.63 0
EBP-FVIII 822 < 0.63 0 823 < 0.63 0 824 1.27 0 825 0.72 0 833
0.74 0 Group 3 AAV2/5-HFN3/ 841 16.785 49.833 EBP-FVIII + Doxil 842
12.425 37.282 (no spikes) 843 13.685 41.466 844 35.225 91.842 845
7.815 12.974 846 24.02 54.853
[0223] Thus, agents that interact with molecules in intracellular
AAV trafficking pathways, such as proteosomes or molecules in the
ubiquitin pathway, by binding to those molecules and/or inhibiting
their activity, are useful to enhance rAAV transduction.
EXAMPLE 4
Proteasome Involvement in rAAV-2 and rAAV-5 Transduction of
Polarized Airway Epithelia In Vitro and In Vivo
[0224] Inhibition of the proteasome with small tripeptide
inhibitors such as LLnL can significantly augment rAAV-2
transduction from the apical membrane of both polarized human
airway epithelia in vitro and mouse lung in vivo (Duan et al.,
2000). As AAV-5 has been reported to have higher tropism for, and
alternate receptors on, the apical membrane of airway epithelia,
increased transduction of airway epithelia from the apical membrane
with rAAV-5 might be due to altered proteasome involvement.
Co-administration of a proteosome modulator and a proteosome
inhibitor was found to augment transduction of both serotypes in a
cell type dependent manner (FIGS. 4-6).
[0225] To better understand serotype-specific differences in airway
transduction, the effect of proteasome inhibitors on rAAV-2 and
rAAV-5 transduction in polarized human airway epithelial cultures
and mouse lung was examined (FIG. 8). A proviral construct
containing 5' and 3' ITRs from AAV-2 flanking a transgene was
packaged into both AAV-2 and AAV-5 capsid to generate AV2.RSVluc
and AV2.RSVlucCap5 viruses which express the luciferase transgene.
rAAV-2, but not rAVV-5, demonstrated a significant difference in
transduction from the apical versus basolateral surface.
Transduction with AV2.RSVluc was 36- and 103-fold greater from the
basolateral membrane at 5 and 14 days post-infection, respectively.
In contrast, AV2.RSVlucCap5 transduced epithelia from the apical
and basolateral membranes with similar efficiencies at both time
points.
[0226] LLnL augments AV2.RSVluc transduction from the apical and
basolateral surfaces. However, application of LLnL selectively
increased AV2.RSVlucCap5 transduction 12-fold only when virus was
applied to the apical surface. These results suggest an interesting
difference in the involvement of the proteasome for various AAV
capsid entry pathways that are effected by cell polarity.
[0227] The proteasome inhibitor Z-LLL was found to induce long-term
(5 month) transduction with rAAV-2 in mouse lung. To determine in
vivo transduction efficiency of AV2.RSVlucCap5, mice were infected
with 6.times.10.sup.10 particles of AV2.RSVlucCap5 by nasal
aspiration alone (control) or in combination with 200 .mu.M Z-LLL,
200 .mu.M doxorubicin or 200 .mu.M Z-LLL and 200 .mu.M doxorubicin
(12 mice per group). Co-administration of Z-LLL induced whole lung
luciferase expression 17.2- and 2.1-fold at 14 (2 weeks) and 42 (6
weeks) days post-infection, respectively (FIG. 9). Interestingly,
luciferase expression was further reduced at 3 months
post-infection (FIG. 10).
[0228] Co-administration of doxorubicin induced whole lung
luciferase expression at levels almost ten times higher than those
for Z-LLL at 2 weeks. Doxorubicin also induced tracheal and bronchi
luciferase expression at higher levels than Z-LLL at 2 weeks. At
six weeks, a similar pattern was observed for Z-LLL and doxorubicin
alone, however, luciferase levels were more than additive in
trachea and bronchi in mice co-administered virus, Z-LLL and
doxorubicin. By three months post-infection, the synergism was no
longer observed. These observations suggest a striking difference
in the kinetics and longevity of induction by Z-LLL between in vivo
studies with rAAV-2 and rAAV-5. Since in vivo transduction is
significantly more efficient with rAAV-5 compared to rAAV-2,
altering proteasome activity may simply enhance the rate of
transduction with rAAV-5. In the case of rAAV-2, this basal rate
may be significantly reduced from the apical membrane in vivo
rendering more sustained augmentation of transduction by proteasome
inhibitors.
[0229] These results also highlight the use of different agents and
vectors to achieve different results. For example, agents and
vectors that result in a steady increase in transgene expression in
particular cells over time may be useful for certain disorders or
conditions while agents and vectors that result in a high burst of
transgene expression may be useful for metabolic disorders such as
hemophilia.
[0230] Ubiquitination and proteasome activity can influence a
myriad of intracellular processes that control protein degradation
and intracellular trafficking. The following examples are designed
to identify the molecular mechanisms of rAAV transduction that are
controlled by the ubiquitin/proteasome system. These studies may
lead to a clearer understanding of pathways and/or molecules that
influence rate-limiting steps in rAAV transduction and can also be
used to identify further useful agents to enhance processing of
rAAV (i.e., endosomal escape, trafficking to the nucleus, and
uncoating) and hence transduction.
EXAMPLE 5
Endosomal Pathways for Serotypes of rAAV
[0231] To delineate the intracellular pathway(s) of rAAV
trafficking in airway epithelial cell lines, the pathway of
intracellular trafficking for type 2 and 5 rAAV is determined using
co-localization techniques with fluorescently-labeled rAAV and
intracellular endosomal markers, biochemical purification of
various endosomal compartments, and inhibition of endosomal
movement using exogenously-expressed specific dominant negative Rab
proteins. Rabs are small GTPases that provide for programmed
delivery of endosomal compartments to various subcellular domains,
and facilitate membrane fusion through GTP-dependent mechanism.
Single cell microinjection of fluorochrome-quenching antibodies was
used to determine the endosomal compartment from which rAAV escapes
based on a color changes of dually-labeled rAAV. rAAV2 and rAAV5
may traffic through multiple endocytic compartments (Late endosome
[LE], golgi, perinuclear recycling endosome [PNRE]), but only one
of these compartments is the point of exit into the cytoplasm.
A. Intracellular Accumulation of rAAV2
[0232] Previous studies have demonstrated that Cy3-labeled rAAV2
co-localizes with FITC-labeled transferrin but not FITC-labeled
Dextran when visualized in Hela cells (Bartlett et al., 2000; Duan
et al., 1999; Sanlioglu et al., 2000). Although virus begins to
accumulate in the nucleus by 1 hour post-infection, a significant
amount accumulates in a perinuclear organization. Transferrin is
known to traffic through the PNRE (also called pericentriolar
recycling endosome), which is an intracellular warehouse for
intracellular sorting of receptors. Thus, rAAV2 may also traffic
through this compartment.
[0233] Rab proteins encompass a group of small GTPases that are
well known for their importance in vesicular sorting and membrane
fusion. Many of these Rab proteins have been extensively
characterized as markers for various intracellular sorting
pathways. GFP-Rab fusion proteins as intracellular markers by which
to compare rAAV2 trafficking to the transferrin sorting pathway
(Rab5.fwdarw.Rab4.fwdarw.Rab11) (Ren et al., 1998; Trischler et
al., 1996).
Methods
[0234] Labeling. rAAV was labeled with a monovalent Cy3
fluorochrome as previously described in Duan et al. (1999) and
Sanlioglu et al. (2000). Typically about 2 fluorochromes label the
rAAV capsid with greater than 85% retention of functional activity.
To facilitate quality control analysis of labeled rAAV, rAAV that
expresses the luciferase genes was used. For all labeling
procedures, rAAV is generated by triple transfection as previously
described in Duan et al. (2001). The labeling procedure was
modified to include a G50 Sephadex gel-filtration step to isolate
virus from free fluorochrome. Fractions were then assessed by slot
blot and functional activity, and peak fractions were then analyzed
by EM.
[0235] Intracellular GFP-tagged endosomal markers. cDNAs that
express N-terminal GFP-tagged proteins that mark various
intracellular compartments including Rab4, 5, 7, 9, and 11
endosomes, golgi, and the proteasome were obtained, as well as
dominant negative constructs for each of these Rabs that prevents
GTP hydrolysis (a function required for endosomal fusion mediated
by each Rab) (Table 3).
TABLE-US-00003 TABLE 3 Protein Endosomal cDNA Source Construct
Expressed Compartment and Use (Ref) Rab5-GFP GFP-tagged Marks Early
Endosome Sonnichsen et al. Rab5a (2000) dnRab5 Rab5a(S34N) Block
Movement out of Li et al. Early Endosome (1993) Rab5-HA HA-tagged
Immunoaffinity Rab5a Isolation of Rab5 Endosomes Rab4-GFP
GFP-tagged Block Movement into Sonnichsen et al. Rab4 the Rab4
compartment (2000) dnRab4 Rab4(S22N) Immunoaffinity Sonnichsen et
al. Isolation of Rab4 (2000) Endosomes Rab4-HA HA-tagged
Immunoaffinity Rab4 Isolation of Rab4 Endosomes Rab11- GFP-tagged
Marks the PNRE Sonnichsen et al. GFP Rab11a (2000) dnRab11
Rab11a(S25N) Block Movement through the PNRE Rab11-HA HA-tagged
Immunoaffinity Rab11a Isolation of Rab11 Endosomes Rab7-GFP
GFP-tagged Marks the Late Bucci et al. Rab7 Endosome to (2000)
Lysosome pathway dnRab7 Rab7(T22N) Block Movement Bucci et al.
through the late (2000) endosome Rab7-HA HA-tagged Immunoaffinity
Rab7 Isolation of Rab7 Endosomes Rab9-GFP GFP-tagged Marks Rab7
Late Barbero et al. Rab9 Endosome to Golgi (2002) Movement dnRab9
Rab(S21N) Blocks endosomal Iversen et al. movement to the golgi
(2001) Rab9-HA HA-tagged Immunoaffinity Rab9 Isolation of Rab9
Endosomes TGN38- GFP-tagged Marks the trans-golgi Girotti et al.
GFP TGN38 network (1996) TGN38- HA-tagged Immunoaffinity HA TGN38
Isolation trans-golgi network LMP2- GFP-tagged Marks the proteasome
Reits et al. GFP LMP2 subunit LMP2 (1997)
[0236] Co-localization of Cy3-rAAV2 with GFP-labeled Rab
compartments. HeLa cells were transfected with GFP-tagged Rab4,
Rab5, and Rab11 expression constructs using standard protocols and
lipofectamine/DNA complexes. At 48 hours following transfection,
HeLa cells were infected with Cy3-rAAV2 at an MOI of 50,000
particles/cell on glass coverslips at 4.degree. C. for 1 hour.
Cells were then washed extensively and either fixed for analysis or
shifted to 37.degree. C. for 1 hour. Samples were then evaluated by
confocal microscopy for the co-localization of Cy3 and GFP
signal.
Results and Conclusions
[0237] GFP-tagged Rab5 and Rab4 show similar patterns of
distribution in HeLa cells at 48 hours post-transfection consistent
with their overlapping distribution within the early endosomal
recycling compartment (Trischler et al., 1999). In contrast,
Rab11-GFP, which marks the PNRE, demonstrated a very unique
distribution within the cell. Co-localization experiments with
Cy3-rAAV2 and Rab11-GFP demonstrate a large percentage of overlap
at 1 hour following infection. However, as expected, no overlap in
signal was detected with bound Cy3-rAAV prior to initiated
endocytosis at 37.degree. C. These findings suggest that rAAV2
traffics through the Rab11 compartment. In HeLa cells, this
compartment predominantly demarcates the PNRE. However, some
overlap exists with trans-golgi as Rab11 has also been shown to
control movement from the PNRE to the golgi. The Golgi-specific
marker TGN-38 is employed to evaluate this possibility. Although a
large extent of overlap between Cy3 and Rab11 signals was observed,
specific intracellular and intranuclear domains contained regions
of no overlap in signal. The intracellular domains are other
potential endosomal compartments through which rAAV2 may migrate
(i.e., late endosome, golgi, lysosome).
B. Localization of rAAV with Rab5 and Rab11
[0238] Endosomal purification techniques were employed to evaluate
the trafficking patterns of rAAV and the effect to which proteasome
inhibitors alter the manner in which virus moves through the
cell.
Methods
[0239] Density gradients. Density gradient centrifugation was used
to isolate mixed populations of endosomes according to their size
and buoyant density. Iodixanol is used as the medium for
fractionation since it can provide an iso-osmotic condition with
low viscosity over a wide range of density.
[0240] Vesicular isolation. Confluent monolayers of IB2, Hela, or
A549 cells grown on one 150 mm dish were incubated with 0.8 mg/ml
biotin-transferrin (Sigma Co., St. Louis, Mo.) or AV2Luc
(MOI=10,000) in prewarmed MEM supplemented with 10 mM Hepes for 30
minutes at 37.degree. C. The cells were harvested by trypsinization
(which removes external, membrane-bound rAAV2 (Duan et al., 2001;
Duan et al., 2000), washed in ice-cold PBS three times, and
harvested into ice-cold homogenization buffer (0.25 M sucrose, 10
mM triethanolamine, 1 mM EDTA, 1 mM PMSF, 100 .mu.g aprotinin).
Cells were then homogenized in a Duall tissue grinder and
centrifuged at 1000.times.g at 4.degree. C. for 10 minutes. The
supernatant, which contains intracellular vesicular compartments
and membranes, but not the nuclei, was designated the post-nuclear
supernatant (PNS). The PNS was subsequently combined with 60%
iodixanol solution to obtain a final concentration of 32% and then
loaded into an SW55Ti centrifuge tube and overlaid with two-step
gradients of 24% and 20% iodixanol. All iodixanol solutions were
prepared in homogenization buffer. Samples were centrifuged at
30,500 rpm for 1 hour at 4.degree. C. Fractions were collected from
the top to bottom of the centrifuge tube at 4.degree. C. (320-500
ul/fraction) since vesicular fractions migrate at the interphase
between 24%/20% iodixanol.
[0241] Western blot analysis. 50 .mu.l of each fraction was loaded
on SDS-PAGE gel and Western blotted for Rab5 (Santa Cruz
Biotechnology), Rab11 (Transduction Laboratories), and/or
biotin-transferrin (Zymed Laboratories). Western blots were
developed using ECL chemiluminescence using HRP conjugated
streptavidin or secondary antibodies.
[0242] TaqMan PCR quantification of viral DNA. PCR primers and the
Taqman probe for AV2Luc DNA quantification were selected using the
Primer Express software program. The forward primer, P1
(5'-TTTTTGAAGCGAAGGTTGTGG-3'; SEQ ID NO:1), and the reverse primer,
P2 (5'-CACACACAGTTCGCCTCTTTG-3'; SEQ ID NO:2), were chosen to
amplify a 32-bp fragment in the promoter region of AV2Luc DNA. The
Taqman probe (5'-ATCTGGATACCGGGAAAACGCTGGGCGTTAAT-3'; SEQ ID NO:3)
was designed following the general rules outlined by the
manufacturer. The Taqman probe carried a 5' reporter dye, 6-carboxy
fluorescein (FAM), and a 3' quencher dye, 6-carboxy tetramethyl
rhodamine, and was synthesized by Genosys. The 25 .mu.l PCR mixture
consisted of 10 .mu.l AV2Luc gradient sample, primers P1 and P2
(final concentration 500 nM), Taqman probe (final concentration 100
nM), and 12.5 .mu.l Taqman Universal Master Mix (PE Applied
Biosystems). For AV2Luc DNA amplification, 1 cycle at 50.degree. C.
for 2 minutes and 1 cycle at 95.degree. C. for 10 minutes were
followed by a two-step PCR procedure consisting of 15 seconds at
95.degree. C. and 1 minutes at 60.degree. C. for 40 cycles.
Amplification, data acquisition, and analysis were performed using
the ABI Prism 7700 Sequence Detector System (PE applied
Biosystems). All standard dilutions of purified AV2Luc, controls,
and samples from the subcellular fractionation were run in
duplicate, and the average value of the copy number was used to
quantify AV2Luc. The standard curve for AV2Luc was accepted when
the slope was between -3.74 and -3.32 and the coefficient of
correlation was >0.990.
Results and Conclusions
[0243] Subcellular fractionation of purified endosomes was shown by
isolating intact endosomes containing preloaded biotin-transferrin.
As expected, transferrin immunoreactivity co-fractionates with the
Rab5 and Rab11 compartments as detected by Western blotting
(fraction 3 and 4). The remaining immunoreactivity in the bottom of
the tube represents lysed endosomes and free Rabs or transferrin in
the PNS. The addition of free biotin-transferrin to the PNS of
unloaded cells does not lead to detectable immunoreactivity in peak
vesicular fractions (data not shown).
[0244] To investigate whether this method could be used to isolate
viral-containing endosomes, similar evaluations were performed on
AV2Luc-infected Hela cells (MOI=10,000). Peak Rab5/Rab11 positive
vesicular fractions (#2-4) co-isolate with internalized rAAV
genomes, following a 30 minute infection at 37.degree. C.
Approximately 50% of rAAV DNA was contained within the endosomal
fraction. There was also a significant portion of rAAV DNA in the
PNS at the bottom of the tube. This likely represents either free
rAAV that has exited the endosome or endosomal lysis during the
processing. However, without the use of more refined methods
proposed in the experimental plan, the interpretation that vector
remaining in the PNS had exited the endosome should be interpreted
cautiously.
C. Use of HA-tagged Rab Proteins for Purification
[0245] Based on a previous report describing the immuno-affinity
isolation of Rab5 and Rab11 endosomal compartments to study
transferrin movement through cells (Trischler et al., 1999), a
novel approach was developed to immuno-isolate numerous endosomal
compartments using HA-tagged Rab marker proteins. These HA-tagged
constructs as described below, partition to the endogenous sites of
their Rab counterparts as well as our ability to immuno-isolate the
Rab5 compartment.
Methods
[0246] N-terminal HA-tagged Rabs were generated by PCR for Rab5,
Rab7, and Rab11 using a forward primer containing the HA epitope. A
CMV-driven plasmid expression construct was employed to express
HA-Rab5 and HA-Rab11 in Hela cells following lipofectamine
transfection. At 72 hours post-transfection, endosomal fractions
were purified and various fractions from the Iodixanol gradient
were evaluated by Western blotting for HA, Rab5, and Rab11. Mixed
populations of endosomes were then used for the immuno-affinity
isolation strategy described below.
[0247] Immuno-affinity isolation of HA-tagged Rab5 endosomal
compartments. Rab5 endosomes were isolated based on a previous
method (Trischler et al., 1999) with modifications. Hela cells were
transfected with HA-Rab5 expression plasmid and peak vesicular
fractions (#3, 4, and 5) were combined from the Iodixanol gradient
and immuno-affinity-purified using Dynabeads M-500 (Dynal Inc)
bound to anti-HA antibodies. Secondary antibody (40 ug anti-rabbit)
was conjugated to Dynabeads (200 .mu.l containing 4.times.10.sup.8
beads/ml) in 0.1 M borate buffer (pH 9.5) for 24 hours at
25.degree. C. with slow rocking. The beads were then placed into
the magnet for 3 minutes to remove the supernatant and washed three
times in 0.1% (w/v) BSA/PBS for 5 minutes at 4.degree. C. A final
wash in 0.2 M Tris (pH 8.5)/BSA was performed for 24 hours.
Finally, the beads were resuspended in BSA/PBS and conjugated to 4
.mu.g primary anti-HA antibody per 107 beads O/N at 4.degree. C.
and washed in BSA/PBS. Vesicular fractions (300 .mu.l) from
2.times.10.sup.7 cells expressing the various HA-tagged Rabs were
mixed with 700 .mu.l coated beads in PBS containing 2 mM EDTA, 5%
BSA, and protease inhibitors. The mixture was then incubated for 6
hours at 4.degree. C. with slow rocking, followed by magnetic
capture and washing in the same tube three times (15 minutes each).
Beads and enriched endosomes were then resuspended in PBS for
Western blotting to assess enrichment of the Rab5 compartment.
Results and Conclusions
[0248] Exogenously-expressed HA-Rab5 and HA-Rab11 partition in an
Iodixanol gradient to fractions typically containing the endosome.
To assess the co-localization of endogenous Rab counterparts with
the exogenously-expressed HA-tagged fusion, Western blots of peak
vesicular fractions using anti-HA, anti-Rab5, and anti-Rab11 were
performed. HA immunoreactivity was only seen in endosomes from
cells transfected with the HA-tagged Rabs. This immunoreactivity
coincided with the peak immunoreactivity for each of the Rab
proteins. These results demonstrated that the tagged Rabs properly
incorporate into endosomes and partition with the endogenous
membrane-bound Rab counterparts.
[0249] Using an immuno-affinity isolation strategy with anti-HA
bound Dynabeads beads, the peak endosomal fraction from
HA-Rab5-transfected Hela cells (#4) was used for Rab5 endosomal
isolation. Immuno-isolation was performed in the presence of
1.degree. anti-HA and 2.degree. anti-rabbit antibodies or with
2.degree. anti-rabbit antibody alone as a control for specificity.
The results demonstrate approximately 30% immuno-isolation of
Rab5-containing vesicles using the HA-Rab5 marker and undetectable
contamination when 2.degree. anti-rabbit antibody was used
alone.
D. Dual Fluorochrome Labeling of rAAV to Follow Endosomal
Escape
[0250] One of the most challenging but important aspects of
intracellular trafficking of rAAV is determining the exact
endosomal compartment from which virions exit into the cytoplasm.
Proteasome inhibitors may modulate this aspect of the rAAV life
cycle by either changing the rate of endosomal escape and/or the
compartment from which rAAV enters into the cytoplasm.
Methods
[0251] To study endosomal escape, single-cell imaging and
microinjection of quenching antibodies against one of two
fluorochromes on a dual-labeled rAAV capsid were performed. The
Alexa Fluor system from Molecular Probes was chosen as a system for
which multiple fluorochromes could be linked to the rAAV capsid at
similar efficiencies. Three dyes (Alexa Fluor.RTM. 488 [green],
Alexa Fluor.RTM. 568 [Red] and Alexa Fluor.RTM. 647 [blue]) were
selected as useful in this regard. Preferably, dual labeling of
rAAV does not change the infection pattern. Also preferably,
microinjection of quenching antibodies against Alexa-488 (Molecular
Probes) can shift fluorescence of dual-labeled rAAV. The general
approach to assess endosomal escape is to inject the cytoplasm of
living cells with anti-Alexa-488 following infection with rAAV that
is dual labeled with Alexa-488 and one of the other dyes.
Alexa-488/568 dual-labeled rAAV, a shift in fluorescence of virus
from yellow to red (i.e., quenching of the green fluorochrome)
indicates movement of virus into the cytoplasm. This approach is
used in combination with GFP-tagged endosomal compartments and/or
dominant negative Rabs to evaluate the compartment from which rAAV
moves into the cytoplasm.
[0252] Alexa labeling of rAAV. The monovalent Alexa succinimidyl
ester reactive dye (Alexa-488 and/or Alexa-568) was dissolved in 50
.mu.l of 1 M bicarbonate. 0.5.times.10.sup.12 particles (determined
by slot blot) of purified AV2Luc in 0.5 ml Hepes buffer was added
to the reaction mixture and incubated for 2 hours. When dual
labeling was performed, equal molar amounts of the two
fluorochromes was used and the reaction time was extended to 3
hours. The labeled rAAV2 was separated from the free dye by
exclusion chromatography. The fractions were tested for infectious
titers on HeLa cells using luciferase assays. The 5 peak fractions
were then combined and used for fluorescent imaging studies.
Imaging studies were performed.
Results and Conclusions
[0253] Assessment of functional particles demonstrated that greater
than 85% activity was retained following label with Alexa dyes
(data not shown). This was similar to results observed with Cy3
labeling. Results from Hela cells infected with Alex-568-labeled
rAAV2 demonstrated a significant overlap in signal with the
GFP-tagged Rab11 compartment. The distribution observed was very
similar to that seen with Cy3-labeled rAAV2. From these studies, it
was concluded that Alexa-labeling of rAAV can be performed, and it
was slightly more sensitive than Cy3-labeling. In these studies,
approximately 3-4 fluorochromes were labeled on each rAAV capsid.
To investigate whether dual labeling procedures could also be
adapted to efficiently label rAAV, studies were conducted that
compared dual Alexa-488/568 and Alexa-568-labeled rAAV2 following a
1 hour infection of Hela cells. These studies, which demonstrate
overlap in the Alexa-488/568 signal, as compared to Alexa-568
alone, confirm that the predominance of rAAV virions are
dual-labeled when both dyes are added to the conjugation
reaction.
[0254] To begin to develop assays for visualizing endosomal release
of rAAV into the cytoplasm, it was determined single cell injection
of Anti-Alexa-488 could quench green fluorescence from dual-labeled
Alexa-488/568 once rAAV entered into the cytoplasm. Results from
these experiments are show in FIG. 17C and depict the fluorescence
of Alexa-488/568 dual-labeled AV2Luc in Hela cells at 2 hour
post-infection following injection with Anti-Alexa-488. Three cells
are shown in the field, of which two were microinjected with
antibody (closed arrowheads). From this study, it is obvious that
the level of Alexa-488 fluorescence is significantly quenched by
injection of anti-Alexa-488 while leaving red channel fluorescence
of Alexa-568 intact. In contrast, fluorescence of both
fluorochromes remains quite high in uninjected cells (open arrow).
The remaining Alexa-488 fluorescence in injected cells is
interpreted as virus still remaining in the endosomal compartment
protected from antibody binding. These findings suggest that a
significant portion of rAAV may be free in the cytoplasm by two hrs
post-infection.
E. Intracellular Trafficking Patterns of rAAV-2 Demonstrate
Significant Cell-Type Specificity
[0255] To further investigate the intracellular mechanisms of
rAAV-2 transduction that might vary between cell types,
immunofluorescent localization of Cy3-rAAV-2 was performed
following transduction of HeLa and IB3 cells. Despite the fact that
rAAV-2 enters these two cell types with similar efficiency, HeLa
cells are much more transducible with rAAV-2 than IB3 cells.
However, IB3 cells demonstrate a much higher responsiveness to
tripeptidyl proteosome inhibitor induction of transduction than
HeLa cells. These differences in transduction may be reflected by
variations in the intracellular trafficking patterns of rAAV-2
between HeLa and IB3 cells.
Methods
[0256] Luciferase-expressing rAAV2 was labeled with Cy3 and
purified by column chromatography. Rab11, Rab7, and Rab9 were
cloned into a pEGFP-C3 vector such that N-terminal EGFP-Rab fusions
were generated. IB3 and HeLa cells were transfected with various
EGFP-tagged Rabs using lipofectamine and infected with Cy3-labeled
rAAV-2 at 4.degree. C. for 30 minutes with an MOI of 10,000
particles/cell. Cells were then washed and shifted to 37.degree. C.
for 30 minutes to 2 hours. Cells were fixed and evaluated by
fluorescent microscopy.
Results
[0257] Fluorescent microscopy was used to evaluate the primary
vesicular compartments in which Cy3-labeled rAAV-2 accumulated
following infection of HeLa and IB3 cells. A substantial degree of
co-localization of Cy3-AAV-2 and EGFP-Rab11 was observed in HeLa
cells from 30 minutes to 2 hours post-infection. This pattern,
however, was not observed in IB3 cells. In contrast, the
Cy3-labeled rAAV-2 was primarily co-localized with EGFP-Rab9 in IB3
cells. In HeLa cells, the degree of co-localization of Cy3-AAV and
EGFP-Rab9 was not predominant. These findings suggest that rAAV-2
traffics through a diversity of intracellular compartments in a
cell type specific manner.
EXAMPLE 6
Altered Trafficking of rAAV
[0258] Proteasome-modulating agents act to increase rAAV
transduction through one or more of the following mechanisms: 1)
increasing the rate at which rAAV accumulates in the primary
compartment through which it emerges to the cytoplasm without
changing the pathway of intracellular trafficking; 2) altering the
pathway of rAAV intracellular trafficking in a manner that leads to
more efficient accumulation in a compartment through which it
emerges to the cytoplasm; 3) increasing the efficiency at which
rAAV breaks out of the endosomal compartment; and/or 4) enhancing
the rate of nuclear trafficking of free rAAV in the cytoplasm.
[0259] Several lines of evidence suggest that proteasome inhibitors
may act to enhance rAAV transduction by increasing the rate of
viral transport to the nucleus (Duan et al., 2000) and/or enhancing
viral processing of the capsid (Yan et al., 2002). First,
proteasome inhibitors such as the tripeptides LLnL and Z-LLL
enhance transduction of both rAAV2 or rAAV5, viruses without
enhancing 1) endocytosis of virus, 2) stability of viral DNA within
the cell, or 3) promoter activity which drives transgene expression
(Duan et al., 2000; Yan et al., 2002). Second, proteasome
inhibitors can be added up to a week following infection of
polarized human airway epithelia and still enhance transduction
(i.e., gene expression). Third, viral capsids for type 2 and type 5
show enhanced ubiquitination in vivo in the presence of proteasome
inhibitors, and purified virus can also be ubiquitinated in vitro
(Yan et al., 2002). Together, these findings strongly suggest that
modulating proteasome activity enhances rAAV transduction for at
least two serotypes and that the mechanism of enhancement involves
some aspect of intracellular viral processing.
A. Proteasome Inhibitors Increase Transport of rAAV2 and rAAV2/5
cell to the Nucleus
[0260] A large number of various classes of proteasome inhibitors
were screened to identify those that had the largest effect. Two
classes of compounds (the tripeptidyl aldehyde LLnL and an
anthracycline derivative doxorubicin), and their ability to induce
rAAV2 and rAAV2/5 transduction in two airway cell lines (IB3 and
A549) are described below.
Methods
[0261] LLnL and Z-LLL are two tripeptidyl aldehydes shown to
inhibit calpains, cathepsins, cysteine proteases as well as the
chymotrypsin-like protease activity of proteasomes (Wagner et al.,
2002; Donkor, 2000; Sasaki et al., 1990). Doxorubicin has also been
shown to inhibit chymotrypsin-like protease activity of proteasomes
(Kiyomiya et al., 2002). Both classes of proteasome inhibitors bind
tightly to the proteasome complex. Dose response curves for these
two proteasome-modulating agents were evaluated on IB3, A549, Hela,
and primary fibroblasts. The responses were consistent for a number
of cell lines and for three different promoters driving luciferase
expression. For one set, CMV-driven luciferase constructs with an
AAV2-based genome were employed that were packaged into AV2 or AV5
capsids. Cells were infected at various doses of AV2Luc and
AV2/5Luc (MOIs 100 to 1000 particles/cell). At the time of
infection, cells were treated with various concentrations of LLnL
or Doxorubicin and gene expression was assayed at 24 hours
post-infection. The effect of proteasome inhibitors on nuclear
uptake of virus was evaluated using a previously-described protocol
for fractionating viral DNA in the cytoplasm and nucleus (Xiao et
al., 2002). Viral DNA content in the cytoplasmic and nuclear
fractions was then evaluated by slot blot hybridization against a
Luciferase DNA probe.
Results and Conclusions
[0262] Results from this analysis demonstrated that both LLnL and
Dox can significantly augment rAAV2 and rAAV2/5 transduction in two
independent airway cell lines (FIG. 11). Although the trends were
similar between these two cell lines and the two serotypes of rAAV,
several features of the induction are worth noting. First,
transduction in IB3 cells was most significantly inducible
(>200-fold) by LLnL, while A549 cells required much higher
concentration of LLnL to achieve 10-fold lower levels of induction.
Hence, IB3 cells appear to be particularly sensitive to LLnL
induction of rAAV. Second, rAAV transduction in both cell lines was
highly inducible (200-fold) by Dox.
[0263] Given previous findings in polarized human airway epithelial
cells that treatment with LLnL increased movement of rAAV to the
nucleus (Duan et al., 2000), it was determined whether LLnL and Dox
treatment at the time of infection also enhanced rAAV movement to
the nucleus. Subcellular fractionation of nuclei and cytoplasmic
extracts from rAAV2-infected IB3 cells, demonstrated that both Dox
and LLnL significantly increased the fraction of viral DNA in the
nuclear compartment (FIGS. 12A and B). These findings suggest that
these two proteasome-modulating agents act to increase rAAV
transduction by mobilizing virus to the nucleus. In summary, these
findings support a growing body of work that the
ubiquitin/proteasome system acts in some manner to control
intracellular processing of rAAV and its movement to the
nucleus.
B. LLnL and Dox Act through Distinct Mechanisms to Modulate the
Proteasome and Enhance rAAV Transduction.
[0264] To test the hypothesis that LLnL and Dox might augment rAAV
transduction through distinct mechanistic interactions with the
proteasome, their effects on rAAV transduction were assessed when
added in combination. If each of these drugs acted to augment
transduction by distinct mechanistic interactions with the
proteasome, then their cumulative effect would be greater than
either individually.
Methods
[0265] Hela, A549, IB3, and primary fetal fibroblasts were
evaluated for AV2Luc and AV2/5Luc transduction in the presence of
LLnL, Dox, or LLnL+Dox at various concentrations. The data shown is
from Hela and A549 cells at the most optimal dose combination that
induces rAV2Luc transduction to a greater extent than each compound
alone.
Results and Conclusions
[0266] Cooperative inhibition of the proteasome by multiple
proteasome inhibitors can provide increased augmentation in rAAV
transduction (FIG. 13). The observation that combined Dox and LLnL
treatment enhances rAAV transduction greater than either compound
alone does not, in and of itself, prove that the mechanisms of
induction are independent of one another. There are several
potential reasons why such drugs might cooperatively enhance rAAV
transduction. First LLnL and/or Dox might alter endosomal routing
of rAAV, enhance endosomal escape, and/or mobilize rAAV in the
cytoplasm to the nuclear pore. Each of these compounds might affect
any one or more of these processes to differing extents and allow
for additive or synergistic affects on rAAV transduction. Hela
cells appear to provide a greater additive effects of Dox and LLnL
on rAAV transduction than A549 cells. Furthermore, it should be
noted that in primary fetal fibroblasts, no additive effect on
transduction is seen (data not shown). In this cell line, Dox most
significantly enhances transduction of rAAV2 and rAAV5, and LLnL
provides no additional induction despite the fact it induced
transduction 10-fold by itself. These interesting cell-specific
differences also imply that certain cellular processes that alter
rAAV transduction may be uniquely controlled by LLnL and Dox
interactions with the proteasome.
EXAMPLE 7
[0267] The mechanism(s) by which proteasome inhibitors augment rAAV
transduction from the apical membrane of airway epithelia may be
reflected in the biologic differences in intracellular trafficking
in apical and basolateral compartments. Co-infection studies from
the apical and basolateral membranes of epithelia with two
different fluorochrome-labeled rAAV viruses were used to directly
visualize how polarity alters intracellular trafficking, as
endocytic pathways from the apical and basolateral membranes of
polarized airway epithelia may differentially utilize the
ubiquitin/proteasome system to modulate vesicular trafficking and
processing of rAAV. Endosomal trafficking pathways for rAAV2 and
rAAV5 identified using cell lines and polarized airway models may
be in vivo using human and mouse bronchial xenograft models with
recombinant adenoviruses expressing either GFP-tagged intracellular
markers or dominant negative Rab proteins.
A. Epithelial Polarity and the Ubiquitin/Proteasome System Uniquely
Affect rAAV transduction from Apical and Basolateral Membranes
[0268] Of particular importance to understanding the intracellular
barriers to rAAV transduction from the apical membrane is an
appreciation of how epithelial polarity alters intracellular
trafficking of virus from the apical and basolateral membranes. It
was previously reported that rAAV2 transduces from the basolateral
membrane of polarized human airway epithelia 200-fold more
effectively then from the apical membrane (Duan et al., 1998).
Interestingly, this reduced transduction from the apical membrane
correlated with the partitioning of high-affinity heparan sulfate
proteoglycan (HSPG) AAV2 receptor to the basolateral membrane, but
did not correlate with a substantial difference in viral
endocytosis from the apical vs basolateral membranes (Duan et al.
1999, Duan et al., 2000). These findings suggest that an
unidentified alternative apical receptor for AAV2 may be present on
the apical surface of human airway epithelia which leads to
endocytosis but also to poor intracellular processing of rAAV2 in
the absence of applied proteasome inhibitors (Duan et al., 2000).
In contrast, rAAV5 has been suggested to infect the apical surface
of airway epithelia more effectively than rAAV2 due to its use of
an alternative receptor that resides on both the apical and
basolateral surfaces (Walters et al., 2001; Zabner et al., 2000).
This finding raises the possibility that the different receptors
for rAAV2 and rAAV5 may also utilize different endosomal processing
pathways.
Methods
[0269] Polarized human airway epithelia were generated as
previously described in Duan et al. (1998), Duan et al. (2000), and
Duan et al. (1998). Both AV2Luc and pseudotyped AV2/5Luc viruses
were utilized in these studies. Infections were performed by
applying equal amounts of virus, in 500 .mu.l of cell culture
media, to the apical or basolateral membrane in the presence of
LLnL (40 .mu.M) for 16 hours as previously described in Duan et al.
(1998). After infection, the epithelia were washed and re-fed with
media lacking LLnL or virus and harvested for luciferase assays at
5 and 14 days post-infection.
Results and Conclusions
[0270] Comparison of AV2Luc and AV2/5Luc transduction from the
apical and basolateral membrane of airway epithelia yielded several
interesting findings (FIG. 14). First, these studies confirmed
previous findings demonstrating a >200 fold higher efficiency of
rAAV2 transducing from the basolateral as compared to the apical
membrane. Second, they also demonstrated a slightly higher level of
transduction from the apical membrane with AV2/5Luc as compared
with AV2Luc, although the difference was not as great as previously
reported by Zabner et al. (2000). Third, only rAAV2 demonstrated a
polarity of infection from the apical and basolateral membranes.
Lastly, only apical transduction for both AAV serotypes was
enhanced by the addition of the proteasome inhibitor LLnL. Thus,
the data suggest a common link between rAAV transduction and the
proteasome independent of the receptor entry pathway.
B. rAAV Gene Conversion is not a Rate-Limiting Step in Transduction
of Polarized Airway Epithelium from the Apical Membrane
[0271] Thus far the data suggests that inhibition of the proteasome
increases the ability of rAAV to migrate to the nucleus and express
its encoded gene. Since rAAV is a single-stranded DNA virus which
packages + or - stands, it must convert its genome to duplex
double-stranded form in order to express encoded transgenes. If
proteasome inhibition also affects this process, the mechanism of
action could be more complicated than proposed. Since increased
nuclear uptake of virus in the presence of proteasome inhibitors
will undoubtedly also increase genome conversion of rAAV,
differentiating between a direct proteasome inhibitor effect on the
level of conversion enzymes and increased nuclear transport of
virus or viral genome, e.g., via cytoskeletal components such as
microtubules or microfilaments.
[0272] To address whether second strand synthesis might also be
rate-limiting in the airway epithelia and enhanced by the
proteasome inhibition, self-complementary AAV vectors (scAAV, also
known as double-stranded AAV or dsAAV) that do not require second
strand synthesis were used. These viruses which contain half-length
genomes (<2.5 kb) have been shown to package either two annealed
single-strand genomes ( i.e., dsAAV) or replication form (Rf)
monomer genomes composed of a covalently joined end ( i.e., scAAV)
(McCarty et al., 2001). Since scAAV vectors have been shown to not
require second strand synthesis to express an encoded transgene,
their onset of gene expression is much more rapid. scAAV vectors
and full-length AAV vectors were employed to demonstrate that
intracellular processing, and not second strand synthesis, is the
primary rate-limiting step in apical transduction of human airway
epithelia.
Methods
[0273] A set of viral vectors was prepared of half-genome length
was prepared. Four GFP-based viruses were generated for this
analysis (AV2eGFP, scAV2eGFP, AV2/5eGFP, and scAV2/5eGFP that had
either 4.7 kb or 2.4 kb length genomes packaged into AAV2 or AAV5
capsids. The data AAV2 capsid viruses were identical to data for
rAAV5 viruses. Functional confirmation of scAAV/dsAAV structure was
performed by analysis of gene conversion and gene expression rates
in Hela cells, sensitivity of transduction in Hela cells to the DNA
synthesis inhibitor hydroxyurea (HU), and by denaturing NaHO gel
electrophoresis. Polarized airway epithelia were infected with the
various vector constructs from the apical membrane in the presence
and absence of applied LLnL at the time of infection. Gene
expression was monitored by quantitative morphometry of GFP
fluorescence at various post-infection time points.
Results and Conclusions
[0274] Evaluation of full-length rAAV and scAAV vectors on Hela
cells demonstrated the previously reported faster rate of onset and
higher levels of gene expression for scAV2eGFP as compared to
AV2eGFP (FIG. 15A) (McCarty et al., 2001). Furthermore,
pretreatment of Hela cells with 5 mM HU significantly decreased
gene expression from AV2eGFP but not scAV2eGFP virus (FIG. 15B).
These findings support the notion that scAAV vectors do not require
DNA synthesis to express encoded transgenes (McCarty et al., 2001).
Furthermore, molecular characterization of Hirt DNA from infected
Hela cells demonstrated a much higher percentage of full-length
scAV2eGFP genomes (2.4 kb) at 24 hours post-infection as compared
to AV2eGFP, which was predominantly single-stranded migrating at
1.6 kb in a native gel (FIG. 15C). Additionally, denaturing NaHO
gel analysis of viral DNA demonstrated that approximately 75% was
Rf (data not shown). Given the Hirt DNA analysis, we assume the
remainder is likely dsAAV. In contrast to the clear enhancement of
gene expression seen with scAV2eGFP vector on Hela cells, results
from analysis of scAV2eGFP and full-length AV2eGFP vector on airway
epithelia demonstrated no discernable difference in apical
transduction in the presence or absence of proteasome inhibitor
(FIGS. 16-17). Although data is only shown for rAAV2 serotypes, the
results were identical for rAAV2/5. These findings strongly suggest
that second strand synthesis is not the major rate-limiting step in
rAAV transduction of human airway epithelia. Additionally, the
finding that LLnL did not alter the profile of expression between
scAV2eGFP and full-length AV2eGFP viruses also suggests that this
proteasome inhibitor does not alter the rate of second strand
synthesis in airway epithelia.
C. Proteasome Inhibitors Enhance the Efficacy of rAAV-Mediated
Functional Correction of CFTR in Polarized Human Airway
Epithelia
[0275] As described below, combined administration of LLnL and Dox
synergistically act to augment rAAV transduction from the apical
membrane of human polarized airway epithelia to a level which can
restore near normal levels of CFTR-mediated chloride current.
Furthermore, analysis of type 5 and type 2 rAAV vectors in mouse
lung using these approaches suggest species-specific differences in
both the synergistic response to proteasome inhibitors and the
optimal AAV serotype when compared to human airway epithelia (FIG.
18).
Methods
[0276] Several vectors were used for this analysis including,
AV2.Luc, AV2/5Luc, scAV2eGFP, AV2LacZdonor, AV2LacZacceptor,
AV2tgCF, AV2/5tgCF, AV2CF83, and AV2/5CF83. AV2LacZdonor and
AV2LacZacceptor virus are two trans-splicing vectors that
reconstitute LacZ expression following intermolecular recombination
and have been previously described in Duan et al. (2001). These
vectors were used to establish the utility of combined proteasome
inhibitor treatment to augment delivery using this approach.
AV2tgCF is the current clinically-used AAV2-based full-length CFTR
vector in which expression of CFTR is driven off the ITR (Aitken et
al., 2001; Wagner et al., 2002). AV2/5tgCF virus has the identical
proviral structure to AV2tgCF but is packaged into AAV5 capsid.
AV2CF83 and AV2/5CF83 viruses have an additional 83 bp minimal
promoter inserted into the AV2tgCF proviral genome to increase gene
expression and are packaged into AAV2 and AAV5 capsids,
respectively. All the CFTR vectors used in the current study were
provided by Target Genetics Incorporated. Infections of polarized
human CF and non-CF airway epithelia were all performed from the
apical membrane at a dose of 10,000 particles/cell for 24 hours in
the presence of Dox and LLnL.
[0277] In vivo Assessment of Gene Transfer in Mouse Lung. In vivo
gene delivery to the lung of BL6 mice was performed by nasal
inhalation of 1.times.10.sup.11 particles of AV2Luc or AV2/5Luc in
the presence of proteasome inhibitor (200 .mu.M Z-LLL and/or 200
.mu.M Dox) as previously described in Duan et al. (2000). For in
vivo studies, it is necessary to use the tripeptide Z-LLL was
employed in place of LLnL because solubility in ethanol is much
higher for Z-LLL. LLnL and Z-LLL perform similarly to augment rAAV
in human polarized airway epithelia (Duan et al., 2000). For in
vivo use, the concentration of tripeptide proteasome inhibitor is 5
to 10-fold higher to augment rAAV transduction and LLnL is
insoluble in ethanol at 200 .mu.M. Mouse lung and tracheas were
harvested separately for analysis at various post-infection time
points and assayed for luciferase activity as previously described
in Duan et al. (2000). Data presented here shows only 14-day time
points for comparison to studies with human polarized airway
epithelia (FIG. 19).
[0278] Functional assays for CFTR complementation. Complementation
of CFTR chloride transport abnormalities in polarized CF airway
epithelia was performed as previously described in Liu et al.
(2002). Short circuit currents of epithelia were measured in Ussing
chambers at 15 days following a 24 hour apical infection in the
presence of Dox and LLnL. CFTR-mediated transport of chloride was
interpreted as the increase in current generated following addition
of 0.1 mM IBMX/10 .mu.M Forskolin to the luminal bath of epithelia
equilibrated with low luminal chloride and 100 .mu.M amiloride. All
CFTR-mediated current was reversibly blocked by the addition of 100
.mu.M bumetanide to the basolateral bath.
Results and Conclusion
[0279] Experiments evaluating the affect of LLnL and/or Dox
treatment of polarized airway epithelia demonstrated a dramatic
synergistic affect on transduction efficiency with rAAV
transduction. As seen in FIG. 15, enhancement of transduction from
the apical surface increased 10 and 100-fold in the presence of
LLnL and Dox, respectively. Remarkably, the combined addition of
Dox and LLnL at the time of infection enhanced transduction
1000-fold with both full-length AV2Luc (FIG. 17A) or
self-complementary scAV2eGFP (FIG. 17B-E). This high level of
augmentation was also capable of facilitating high level dual
vector trans-splicing reconstitution of LacZ (FIG. 17F). Expression
of the reconstituted LacZ gene product was only seen in epithelia
co-infected with both AV2LacZdonor and AV2LacZacceptor viruses.
[0280] CFTR complementation studies in CF polarized airway
epithelia using CFTR rAAV vectors, which compared both AAV2 and
AAV5 capsid-mediated transduction in the presence and absence of
optimal proteasome inhibitor combinations (Dox and LLnL),
demonstrated several interesting findings (FIG. 19). First, it was
evident that rAAV2 capsid vectors performed as well or slightly
better than rAAV5 in the absence of proteasome inhibitor. These
findings are similar to those previously discussed using luciferase
vectors (FIG. 19) but differ from one previous report (Zabner et
al., 2000). Second, as seen with luciferase-based vectors,
rAAV2-mediated CFTR delivery performed dramatically better in the
presence of proteasome inhibitors than that seen with rAAV5. These
findings suggest that in the presence of proteasome inhibitors,
rAAV2 capsid-based vectors are perhaps the better vector for gene
therapy of CF. Third, there was a tangible increase in correction
seen with the AV2CF83 minimal promoter as compared to the current
clinical vector AV2tgCF that utilizes the ITR as a promoter.
Cumulatively, these findings demonstrate the need to circumvent
intracellular barriers by modulating the proteasome to achieve
functional expression of CFTR and support the current clinical
observations with AV2tgCF (also called tgAAVCF) that substantial
vector DNA can be found in airway epithelia without RT PCR
detectable mRNA (Aitken et al., 2001). Thus far, results have
demonstrated a direct correlation of CFTR functional correction
with mRNA expression from the vectors.
[0281] Studies comparing transduction of rAAV2 and rAAV2/5 vectors
and the effect of Dox and Z-LLL on transduction in mouse lung have
demonstrated several notable differences to those seen in human
polarized airway model. First, AV2/5 vectors perform substantially
better (100-fold) in mouse lung and trachea as compared to AV2
vectors. This finding in mice supports several other reports in the
field comparing AV2 to AV2/5 (Aurrichio et al., 2002; Zabner et
al., 2000), but is notably different than observations in polarized
human airway epithelia that demonstrate near equivalent
transduction with these two serotypes. Second, Z-LLL and Dox both
substantially increased transgene expression from AV2/5 vectors to
a level of 10 and 100-fold induction, respectively. Third, the lack
of synergism in the induction of AV2/5 vectors when both Dox and
Z-LLL were given at the time of infection. In fact, the combination
of the two drugs appeared to inhibit overall transduction (FIG.
19).
[0282] These differences between rAV2 and rAV2/5 transduction in
mouse and human airways are relevant to evaluating mechanisms of
proteasome involvement in rAAV transduction in the airway for
several reasons. First, the mouse is extensively used as a
preclinical model and knowledge about differences in transduction
biology between humans is made. Second, tripeptidyl aldehyde (i.e.,
LLnL and Z-LLL) and anthracycline derivatives (i.e., Dox) may
enhance intracellular processing of rAAV through overlapping yet
distinct mechanisms. Hence, the lack of synergism in the induction
of rAAV transduction in the mouse airway may provide clues as to
the mechanism of action of these compounds.
EXAMPLE 8
In Vitro and In Vivo Activities of Additional Proteasome
Modulators
[0283] Based on results with doxorubicin, a small number of FDA
approved anthracyclines were tested for their relative in vitro and
in vivo activities on AAV transduction. HeLa cells were infected
with 100 ppc AAV2FLAG-Luc for 2 hours in the presence of different
anthracyclines, e.g., doxorubicin, daunarubicin (Cerubidine),
epirubicin (Ellence.TM.), and idarubicin (Idamycin.RTM.), and cells
harvested 48 hours later. The anthracyclines were pharmaceutical
grade, and prepared according to the manufacturer's instructions.
Prior to use, the agents were diluted in sterile water to an equal
mass, e.g., 0.6 .mu.g/mL, 3 .mu.g/mL and 6 .mu.g/mL. The results
are shown in FIG. 20. For example, 3 .mu.g/mL idamycin increased
luciferase expression by over 5000-fold while doxorubin increased
luciferase expression by 58-fold. Generally, the potency was as
follows:
idarubicin>daunarubicin>epirubicin>doxorubicin.
[0284] Six groups of ten, five-to-seven week-old, Balb/c mice (5
male and 5 female per group) were employed in a comparison of the
relative in vivo potency and safety of different anthracycline
derivatives at a single dose after intranasal delivery. Treatment
was administered as shown in Table 4. Animals were followed for
seven days post dose.
TABLE-US-00004 TABLE 4 rAAV Proteasome Treatment Modulator Route of
(Dose in Proteasome Dose Administration Day of Group R.sub.x DRP)
Modulator (% of HDE) (rAAV/Inhibitor) Sacrifice 1 No R.sub.x
Vehicle Vehicle 0 Intranasal/ 7 control Intranasal 2 Vector 1
.times. 10.sup.12 Vehicle 0 Intranasal/ 7 control AAV2-GFP +
Intranasal 1 .times. 10.sup.11 AAV2-Luc 3 Test 1 1 .times.
10.sup.12 Doxorubicin 10 Intranasal/ 7 AAV2-GFP + Intranasal 1
.times. 10.sup.11 AAV2-Luc 4 Test 2 1 .times. 10.sup.12 Idamycin 10
Intranasal/ 7 AAV2-GFP + Intranasal 1 .times. 10.sup.11 AAV2-Luc 5
Test 3 1 .times. 10.sup.12 Doxil 10 Intranasal/ 7 AAV2-GFP +
Intranasal 1 .times. 10.sup.11 AAV2-Luc 6 Positive 1 .times.
10.sup.12 Doxil 75 Intranasal/ 7 control AAV2-GFP + Intravenous 1
.times. 10.sup.11 AAV2-Luc
[0285] The dose of modulator was based on the Human Dose Equivalent
(HDE) and is summarized below in Table 5. For intranasal dose
administration, the dose was held constant at 10% of the HDE. For
the intravenous positive control (Doxil), a dose of 10 mg/kg (75%)
of the HDE was used. This represented the lowest dose that gave a
10% increase in mean and median luciferase expression in earlier
studies.
TABLE-US-00005 TABLE 5 Human dose equivalent calculations 10% of
Human 10% of human Drug Human 10% Human dose in mg/kg dose per 20
Volume of Concentration dose Dose for a mouse gram mouse stock drug
Drug (mg/mL) (mg/m.sup.2) (mg/m.sup.2) (dose mg/m.sup.2/3) (mg)
(mL) per mouse Adriamycin 2 40-75 7.5 2.5 mg/kg 0.05 mg 0.025 mL
Idamycin 1 10-12 1.2 0.4 mg/kg 0.008 mg 0.008 mL Doxil 2 10-40 4.0
1.3 mg/kg 0.026 mg 0.013 mL Dose calculation: Animal (mouse) dose
in mg/kg .times. 3 (mouse km) = dose in mg/m.sup.2. mg per mouse =
Dose in mg/kg .times. 0.02 kg mouse
[0286] Safety endpoints included morbidity and mortality, clinical
observations, body weights, gross necropsy observations and
histopathology. Transduction endpoints included luciferase and GFP
analysis.
[0287] On the day of sacrifice, the left lung was clamped off at
the level of the extrapulmonary bronchi, removed and frozen on dry
ice. The left lung was homogenized and processed for luciferase
expression using Promega's luciferase assay system (Madison, Wis.).
Luminescence was measured using the Berthold AutoLumat LB953
instrument. Samples were normalized for total protein using
Pierce's Coomassie Plus Protein Assay Reagent (Rockford, Ill.).
[0288] Intranasal administration of doxorubicin and idamycin at 10%
HDE were both associated with early mortality of some animals,
ruffled hair coats and sick mice. In addition, those animals that
survived also lost considerable body weight over the week. The
intranasally doxil treated mice did better than the doxorubicin- or
idamycin-treated animals in that there was no early mortality and
they appeared clinically normal. However, they also lost weight.
The intravenously doxil treated mice fared the best.
[0289] Intranasal treatment of doxorubicin and idamycin resulted in
increased luciferase expression (FIG. 21 and Table 6). Treatment
with doxil at a 10% HDE (both intravenously and intranasally)
resulted in an average increase in luciferase expression by 49- and
74-fold, respectively, 7 days post-dose.
TABLE-US-00006 TABLE 6 Fold increase in luciferase expression
Standard Fold Rx Average Deviation Increase Vehicle - M 1.28E+03
4.05E+02 Vehicle - F 1.32E+03 6.64E+02 Vehicle 1.30E+03 5.19E+02 No
Rx - M 7.28E+03 5.01E+03 1 No Rx - F 3.56E+03 1.27E+03 1 No Rx
5.63E+03 4.12E+03 1 Doxorubicin (10% HDE) - M *4.21E+06 *2.06E+06
578 Doxorubicin (10% HDE) - F 5.44E+05 4.00E+05 153 Doxorubicin
(10% HDE) 1.77E+06 2.13E+06 314 Idamycin (10% HDE) - M 8.11E+05
2.81E+05 111 Idamycin (10% HDE) - F 2.02E+05 1.05E+05 57 Idamycin
(10% HDE) 5.06E+05 3.80E+05 90 Doxil (10% HDE) - M 6.68E+05
2.57E+05 92 Doxil (10% HDE) - F 1.65E+05 7.15E+04 46 Doxil (10%
HDE) 4.16E+05 3.19E+05 74 Doxil (75% HDE iv) - M 3.16E+05 2.69E+05
43 Doxil (75% HDE iv) - F 2.31E+05 1.21E+05 65 Doxil (75% HDE iv)
2.73E+05 2.02E+05 49 *Average and standard deviation were
calculated from two numbers
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[0503] All publications, patents and patent applications are
incorporated herein by reference. While in the foregoing
specification, this invention has been described in relation to
certain preferred embodiments thereof, and many details have been
set forth for purposes of illustration, it will be apparent to
those skilled in the art that the invention is susceptible to
additional embodiments and that certain of the details herein may
be varied considerably without departing from the basic principles
of the invention.
Sequence CWU 1
1
3121DNAArtificial SequenceA synthetic oligonucleotide 1tttttgaagc
gaaggttgtg g 21221DNAArtificial SequenceA synthetic oligonucleotide
2cacacacagt tcgcctcttt g 21332DNAArtificial SequenceA synthetic
oligonucleotide 3atctggatac cgggaaaacg ctgggcgtta at 32
* * * * *