U.S. patent application number 10/099252 was filed with the patent office on 2002-09-19 for replication-defective recombinant aav virions.
This patent application is currently assigned to Chiron Corporation. Invention is credited to Dwarki, Varavani, Escobedo, Jaime, Ladner, Martha Baillie, Zhou, Shang-Zhen.
Application Number | 20020132336 10/099252 |
Document ID | / |
Family ID | 26732952 |
Filed Date | 2002-09-19 |
United States Patent
Application |
20020132336 |
Kind Code |
A1 |
Dwarki, Varavani ; et
al. |
September 19, 2002 |
Replication-defective recombinant AAV virions
Abstract
The invention provides a method for producing purified
replication-defective recombinant AAV virions. The method comprises
introducing into a suitable host cell an AAV vector, an AAV helper
construct and an adenoplasmid accessory construct into the host
cell. The adenoplasmid accessory plasmid is composed adenovirus
plasmid DNA unable to be packaged into adenoviral particles because
it lacks packaging signal sequence(s) or it contains additional
sequences making it too large to package. The host cell is cultured
to produce crude rAAV virions and then lysed. The resulting cell
lysate is applied to a chromatographic column containing sulfonated
cellulose or subjected to cesium chloride equilibrium gradient
centrifugation and the purified rAAV virions are recovered.
Inventors: |
Dwarki, Varavani; (Alameda,
CA) ; Ladner, Martha Baillie; (Oakland, CA) ;
Escobedo, Jaime; (Alamo, CA) ; Zhou, Shang-Zhen;
(Alameda, CA) |
Correspondence
Address: |
ALISA HARBIN, ESQ.
CHIRON CORPORATION
INTELLECTUAL PROPERTY - R440
P.O. BOX 8097
EMERYVILLE
CA
94662-8097
US
|
Assignee: |
Chiron Corporation
4560 Horton Street
Emeryville
CA
|
Family ID: |
26732952 |
Appl. No.: |
10/099252 |
Filed: |
March 15, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10099252 |
Mar 15, 2002 |
|
|
|
09841768 |
Apr 24, 2001 |
|
|
|
09841768 |
Apr 24, 2001 |
|
|
|
09127268 |
Jul 31, 1998 |
|
|
|
6221646 |
|
|
|
|
60054371 |
Jul 31, 1997 |
|
|
|
Current U.S.
Class: |
435/235.1 ;
435/239; 435/320.1; 435/456; 435/69.1; 536/23.1 |
Current CPC
Class: |
C12N 2750/14143
20130101; C12N 2710/10343 20130101; C12N 7/00 20130101; C12N 15/86
20130101; C12N 2750/14162 20130101 |
Class at
Publication: |
435/235.1 ;
435/456; 435/239; 435/320.1; 435/69.1; 536/23.1 |
International
Class: |
C12N 007/00; C12N
015/861; C07H 021/02; C12P 021/06; C12N 007/02; C12N 015/09; C12N
015/70; C12N 015/86; C07H 021/04; C12N 007/01; C12N 015/00; C12N
015/63; C12N 015/74 |
Claims
What is claimed is:
1. A method for producing replication-defective recombinant AAV
virions substantially free of wild-type AAV and helper adenovirus,
comprising: a. introducing into a suitable host cell (i) an AAV
vector that is free of AAV coding sequences and that comprises a
heterologous gene operatively positioned between two AAV ITRs, (ii)
an AAV helper construct having at least one gene encoding an AAV
capsid protein, and (iii) an adenoplasmid accessory construct
having a full adenoviral genome that either lacks a packaging
signal or that contains sufficient additional nucleotides to be
rendered unpackagable, to produce a transformed host cell; b.
culturing the transformed host cell to produce
replication-defective recombinant AAV virions having said
heterologous gene; and c. lysing the cultured host cell to obtain
said replication-defective recombinant AAV virions substantially
free of wild-type AAV and adenovirus particles.
2. The method of claim 1, wherein the adenoplasmid accessory
construct has a full adenoviral genome that lacks a packaging
signal.
3. The method of claim 1, wherein the adenoplasmid accessory
construct having a full adenoviral genome that contains sufficient
additional nucleotides to be rendered unpackagable.
4. The method of claim 1, wherein the heterologous gene encodes a
human protein.
5. The method of claim 4, wherein said human protein is
erythropoietin, thrombopoietin (G-CSF), Factor VIII, Factor IX,
Factor Xa, human growth hormone, leptin or IL-2.
6. The method of claim 1, wherein the adenoplasmid accessory
construct is pJM17, pBHG10 or pBHG11.
7. The method of claim 1 further comprising the steps of: d.
applying the lysate of step (c) to a column comprising sulfonated
cellulose; and e. recovering purified replication-defective
recombinant AAV virions substantially free of host cell proteins
and host cell debris.
8. The method of claim 2, wherein the heterologous gene encodes a
human protein.
9. The method of claim 8, wherein said human protein is
erythropoietin, thrombopoietin (G-CSF), Factor VIII, Factor IX,
Factor Xa, human growth hormone, leptin or IL-2.
10. The method of claim 2, wherein the adenoplasmid accessory
construct is pJM17, pBHG10 or pBHG11.
11. The method of claim 2 further comprising the steps of: d.
applying the lysate of step (c) to a column comprising sulfonated
cellulose; and e. recovering purified replication-defective
recombinant AAV virions substantially free of host cell proteins
and host cell debris.
12. The method of claim 3, wherein the heterologous gene encodes a
human protein.
13. The method of claim 12, wherein said human protein is
erythropoietin, thrombopoietin (G-CSF), Factor VIII, Factor IX,
Factor Xa, human growth hormone, leptin or IL-2.
14. The method of claim 3, wherein the adenoplasmid accessory
construct is pJM17, pBHG10 or pBHG11.
15. The method of claim 3 further comprising the steps of: d.
applying the lysate of step (c) to a column comprising sulfonated
cellulose; and e. recovering purified replication-defective
recombinant AAV virions substantially free of host cell proteins
and host cell debris.
16. The method of claim 1, wherein in said two AAV ITRs are
wild-type.
17. The method of claim 16, wherein said two AAV ITRs are AAV-2
ITRs.
Description
[0001] This application is related provisional application S. No.
60/054,371, filed Jul. 31, 1997, from which priority is claimed
under 37 C.F.R. .sctn. 119 and which is incorporated herein by
reference in its entirety.
BACKGROUND
[0002] A. Field of the Invention
[0003] The present invention is directed to a method for the
production of recombinant AAV virions containing a gene of
interest. More particularly, the present invention is directed to a
method for producing rAAV virions free of wild-type type AAV and
helper virus. The present invention is useful because it produces a
highly pure rAAV virion suitable for evaluation of gene therapy
protocols and/or use in gene therapy.
[0004] B. Background of the Invention
[0005] Adeno-associated virus (AAV) is a non-pathogenic,
replication-defective parvovirus that has a biphasic life cycle. In
the absence of a helper virus, the AAV genome integrates into the
hot cell's genome to establish a latent infection. In the presence
of a helper virus, such as adenovirus, herpes simplex virus or
vaccinia virus, the AAV genome is rescued from latency and is
reproduced to establish a lytic infection. See Muzyczka, Curr.
Topics Microbiol Immunol. 158 (1992) 97-129; Bems and Linden,
BioEssays 17 (1995) 237-245. The helper virus is known to provide
the functions needed for AAV replication. In the absence of helper
virus, AAV stably integrates into human chromosome 19 site
specifically. See Kotin, Proc. Natl. Acad. Sci. 87 (1990) 2211-15;
Samulski, EMBO J 10 (1991) 3941-50. The AAV genome consists of a
4.7 kb linear, single-stranded, DNA molecule with 145 bp inverted
terminal repeats at each end. The remaining, non-repeated sequences
encode for the viral proteins, called rep and cap, involved in
virus replication and packaging. The AAV ITRs are the only cis
elements required for the viral replication, packaging and
integration; the rep and cap functions can be provided in trans.
See McLaughlin, J. Virol. 62 (1988) 1963-73; Samulski J. Virol. 63
(1989) 3822-28.
[0006] Recombinant AAV (rAAV) vectors are attractive vehicles for
human gene therapy because the vectors do not require AAV coding
sequences to be expressed viral coding sequences, the viruses
(viral particles) is capable of infecting non-dividing and dividing
cells efficiently, it has a broad host range and the virions have
high physical stability. See Carter, Curr. Opin. Biotech 3 (1992)
533-39; Bachman, Intervirology 11 (1979) 248-54. The most widely
used method of generating rAAV particles is called the
invention/transfection method. This method involves transfection of
host cells, typically 293 cells, with AAV vector plasmid (i.e.,
plasmid carrying the gene of interest bounded by the AAV ITRs) and
with helper plasmid (i.e., plasmid providing the AAV helper
functions rep and cap but lacking the ITRs) and infection with
adenovirus or herpes virus. See McCown, Brain Res. 713 (1996)
99-107; McLaughlin, J. Virol 62 (1988) 1963-73. In this standard
method, the helper virus can be separated from AAV vectors by
density gradient centrifugation and any residual infectious helper
virus inactivated by heat. However, the resulting AAV vector
preparations still may contain low levels of infectious helper
virus and proteins that may contribute to the immunogenicity of the
composition and present a potential hazard for human
administration. Also, the helper virus is a pathogenic virus and
poses a health risk to laboratory personnel involved in the
manufacturing process. Moreover, the large amount of helper virus
particles and proteins generated during the infection process makes
it difficult to achieve high levels of purity. Heat treatment can
inactivate infectious adenovirus, but the treatment leads to a
3040% drop in the tier of functional rAAV virions and it has been
difficult to remove all of the adenoviral proteins, even by
multiple rounds of CsC1 gradient purifications.
[0007] Recently, there have been reports of rAAV production using
cell lines providing the necessary helper function for rAAV
packaging. See for example, Clark, Gene Therapy 3 (1996) 1124-32;
Chiorini, Human Gene Therapy 6 (1995) 153142; Clark, Gene Therapy 6
(1995) 132941, Flotte Gene Therapy 2 (1995) 29-37 and Tamayose,
Human Gene Therapy 7 (1995) 507-13. this method likewise leads to
the generation of infectious adenovirus or herpes virus, which must
be purified away from the rAAV particles. Although rAAV particles
can be purified on CsC1 gradients, often the final preparations are
contaminated with adenovirus. Also, CsC1 gradient centrifugation
protocol is cumbersome to adapt to large-scale manufacturing.
[0008] There are several studies employing adenovirus mutants
delineating the role of various regions of adenovirus (helper
virus) necessary for AAV production. There is evidence to
demonstrate that the adenoviral DNA replication genes, E2b, E3 and
several adenoviral late genes are not required for AAV replication.
See Laughlin, J. Virol. 41 1982) 868-876; Myers, J. Virol. 35
(1980) 65-75; Jay, Proc. Natl. Acad. Sci. 78 (1981) 2927-31;
Carter, Virology 126 (1983) 505-16; Ito and Sazuki, J. Gen. Virol.
9 (1970) 243-45; Strauss, J. Virol. 17 (1975) 14048; and Janik,
Virology 168 (1989) 320-29. From these results, it can be
predicated that the "accessory" functions that may be necessary to
support AAV replication include adenoproteins E2a and E4, as well
as VA I RNA. An alternative method of producing rAAV is disclosed
in PCT Patent Publication WO 97/17458, published May 15, 1997. In
that document, the accessory functions capable of supporting rAAV
virion production are provided in the from of one or more vectors
containing the adenovirus VA sequence, the adenovirus E4 ORF6
coding region and/or the adenovirus E2a 72 kD coding region.
However, upon production of the rAAV virions, numerous adenoviral
proteins encoded by the foregoing sequences are produced and must
be removed. Moreover, the production of rAAV using the helper
vectors described in WO 97/17458 is undesirable because it requires
periodic auditing to verify the continued presence and operability
of the vectors. Accordingly, there remains a need in the art to
provide a system capable of producing commercially significant
levels of rAAV virions simply and efficiently.
SUMMARY OF THE INVENTION
[0009] The present invention is directed to a method for producing
replication-defective AAV virions that avoids the production of
live adenovirus, the necessity of monitoring for multiple
adenoviral accessory functions, and the need for cumbersome
purification protocols. More particularly, the present invention is
directed to a method for producing replication-defective
recombinant AAV virions substantially free of wild-type AAV and
helper adenovirus, comprising:
[0010] a. introducing into a suitable host cell (i) an AAV vector
that is free of AAV coding sequences and that comprises a
heterologous gene operatively positioned between two AAV ITRs, (ii)
a replication-defective AAV helper construct having at least one
gene encoding an AAV capsid protein, and (iii) an adenoplasmid
accessory construct having a full adenoviral genome that either
lacks a packaging signal or that contains sufficient additional
nucleotides to be rendered unpackagable, to produce a transformed
host cell;
[0011] b. culturing the transformed host cell to produce
replication-defective recombinant AAV virions having the
heterologous gene; and
[0012] c. lysing the cultured host cell to obtain
replication-defective recombinant AAV virions substantially free of
wild-type AAV and adenovirus particles.
[0013] The host cells that are suitable for use in the method of
the present invention are mammalian host cells, preferably human
host cells.
[0014] In another aspect, the invention is directed to a method of
producing purified recombinant AAV virions, comprising:
[0015] a. introducing into a suitable host cell (i) an AAV vector
that is free of AAV coding sequences and that comprises a
heterologous gene operatively positioned between two AAV ITRs, (ii)
a replication-defective AAV helper construct having at least one
gene encoding an AAV capsid protein, and (iii) an adenoplasmid
accessory construct having a full adenoviral genome that either
lacks a packaging signal or that contains sufficient additional
nucleotides to be rendered unpackagable, to produce a transformed
host cell;
[0016] b. culturing the transformed host cell to produce
replication-defective recombinant AAV virions having the
heterologous gene;
[0017] c. lysing the cultured host cell to obtain
replication-defective recombinant AAV virions substantially free of
wild-type AAV and adenovirus particles;
[0018] d. applying the lysate of step (c) to a column comprising
sulfonated cellulose; and
[0019] e. recovering purified replication-defective recombinant AAV
virions substantially free of host cell proteins and host cell
debris.
[0020] Alternatively, the lysate from step (c) is subjected to
cesium chloride equilibrium gradient centrifugation, and the
purified replication-defective rAAV virions containing the
heterologous gene are recovered. The AAV vector, the
replication-defective AAV helper construct and the adenoplasmid
accessory construct are combined either simultaneously or
sequentially.
[0021] The advantage of the triple transfection protocol utilized
in the methods of the present invention is the significant purity
of rAAV preparations after CsC1 gradient purification. The Western
plot analysis shows that the triple transfection method, while
producing equivalent amounts of rAAV particles as the standard
transfection/infection protocol, results in much lower adenovirus
protein production in both the initial lysates and in the final
purified product. This demonstrates the advantage of the triple
transfection protocol over the standard prior art
transfection/infection protocol, in which there is a significant
amplification of adenovirus in the culture and robust adenovirus
gene expression. In addition, because the adenoplasmid accessory
construct used in the methods the this example lacks a packaging
signal, there are no adenovirus particles in the purified material.
This protocol also eliminates the health and safety concerns raised
by the use of live adenovirus and allows production of rAAV
particles in a safe manner. Importantly, the method simplifies the
downstream purification process, thereby enabling relatively
efficient and economical large-scale manufacturing.
DETAILED DESCRIPTION OF THE INVENTION
[0022] In its first aspect, the present invention is directed to a
method for producing replication-defective recombinant AAV virions
substantially free of wild-type AAV and helper adenovirus,
comprising:
[0023] a. introducing into a suitable host cell (i) an AAV vector
that is free of AAV coding sequences and that comprises a
heterologous gene operatively positioned between two AAV ITRs, (ii)
a replication defective AAV helper construct having at least one
gene encoding an AAV capsid protein, and (iii) an adenoplasmid
accessory construct having a full adenoviral genome that either
lacks a packaging signal or that contains sufficient additional
nucleotides to be rendered unpackagable, to produce a transformed
host cell;
[0024] b. culturing the transformed host cell to produce
replication-defective recombinant AAV virions having the
heterologous gene; and
[0025] c. lysing the cultured host cell to obtain
replication-defective recombinant AAV virions substantially free of
wild-type AAV and adenovirus particles.
[0026] It is within the scope of the present invention that the
above described method further comprise the steps of:
[0027] d. applying the lysate of step (c) to a column comprising
sulfonated cellulose; and
[0028] e. recovering purified replication-defective recombinant AAV
virions substantially free of host cell proteins and host cell
debris.
[0029] The host cells that are suitable for use in the method of
the present invention are mammalian host cells, preferably human
host cells.
[0030] The AAV vector, replication-defective AAV helper construct
and adenoplasmid accessory construct of step (a) of the method of
the present invention are prepared using conventional methods of
virology, molecular biology, microbiology and recombinant DNA
techniques. Such techniques are well known and explained fully in
the literature, including, for example, in Sambrook, Molecular
Cloning: A Laboratory Manual (Current Ed.); DNA Cloning: A
Practical Approach (D. Glover, ed.); Oligonucleotide Synthesis
(Current Ed., N. Gait, ed.); Nucleic Acid Hybridization (Current
Ed., B. Hames and S. Higgins, eds.); Transcription and Translation
(Current Ed., B. Hames and S. Higgins, eds.); CRC Handbook of
Parvoviruses (P. Tijessen, ed.); Fundamental Virology, 2d Edition
(B. N. Fields and D. M. Knipe, eds.); Current Protocols in Human
Genetics, Vol. 1 (N. Dracopoli, ed.). These publications and all
other publications referenced throughout this specification are
expressly incorporated herein by reference.
[0031] Gene transfer, gene therapy or gene delivery refer to
methods, techniques or systems for reliably inserting into a host
cell a heterologous or a foreign DNA or a DNA not normally
expressed. The resultant insertion can be by integration of
transferred genetic material into the host cell genomic DNA, by
extrachromosomal replication and expression of transferred
replicons or in a non-integrated manner.
[0032] Vector means any genetic element that is capable of
replication when associated with the proper control elements and
that can transfer DNA or RNA sequences between cells. Examples
include plasmids, phages, transposons, cosmids, chromosomes,
viruses, and virions and include cloning and expression vehicles
and viral vectors.
[0033] The replication-defective AAV virions produced by the method
of the present invention comprise a gene (DNA) encoding a
therapeutic protein operably positioned between a pair of
adeno-associated virus inverted terminal repeats ("AAV ITRs"). AAV
ITRs are art-recognized regions found at each end of the AAV genome
that function together in cis as recognition signals for DNA
replication and for packaging the AAV vector into an AAV coat. The
nucleotide sequences of the AAV ITR regions for the various AAV
serotypes (i.e., AAV-1 to AAV-7) are known in the art and vary in
size with the serotpe. Typically, the AAV ITRs range in size from
about 125-145 bp. See for example, Kotin, Human Gene Therapy 5
(1994) 693-801 and Berns "Parvoviridae and their Replication" in
Fundamental Virology, 2d Edition (B. N. Fields and D. M. Knipe,
eds.). As used here, the AAV ITRs of Applicants' recombinant
replication-defective retrovirion need not be identical to the
nucleotide sequence of the native, i.e., wild-type, sequence, but
may be altered by insertion, deletion or substitution of
nucleotides. Further, the two AAV ITRs may be derived from any of
the AAV serotypes, for example AAV-1, AAV-2, AAV-3, AAV-4, AAV-5
and AAV-7, and need not be identical to or derived from the same
serotype, so long as they permit integration of the heterologous
sequence of interest into the recipient cell genome when AAV rep
gene products are present in the cell.
[0034] The AAV rep coding region is the art-recognized region of
the AAV genome that encodes the proteins required for replication
of the viral genome and for insertion of the viral genome into a
host genome during latent infection. The rep coding region includes
at least the four genes encoding the two long forms of rep (rep 78
and rep 68) and the two short forms of rep (rep 52 and rep 40). For
more details see, for example, Muzyczka, Current Topics in
Microbiol. 158 (1992) 97-129 and Kotin, Human Gene Therapy 5 (1994)
793-801. The rep coding region may be derived from any AAV serotype
or from a functional homologue such as the human herpes virus 6 rep
gene. The region need not include all of the native sequence, but
may be altered by insertion, deletion or substitution of
nucleotides, so long as the sequence that is present provides for
sufficient integration when expressed in a suitable recipient cell.
Preferably, the AAV vector and virions utilized in the present
invention lack one or more of the rep proteins so as to render it
replication-defective. More preferably, the AAV vector of the
present invention lacks all four of the rep proteins.
[0035] The AAV cap coding region is the art-recognized region of
the AAV genome that encodes the capsid or coat proteins, VP1, VP2
and VP3, that package the viral genome. For more details, see, for
example, Muzyczka, Current Topics in Microbiol. 158 (1992) 97-129
and Kotin, Human Gene Therapy 5 (1994) 793-801. The cap coding
region may be derived from any AAV serotype or from a functional
homologue. The cap coding region may be altered by insertion,
deletion or substitution of nucleotides, so long as the sequence
present provide for sufficient packaging when expressed in a
suitable recipient cell. Although the cap coding region is
preferably not included in the AAV vectors and the
replication-defective AAV virions employed in the present
invention, it needs to be included in a helper vector that is
expressed in a packaging cell that recognizes and packages the ITRs
and the gene(s) positioned therebetween.
[0036] Thus, the term "AAV vector," as used herein means a vector
derived from an adeno-associated virus serotype that includes at
least those sequences required in cis for replication and
packaging, for example, a pair of functional ITRs flanking a
heterologous (i.e., non-AAV) nucleotide sequence. With this
criterion, any AAV vector of any serotype can be employed in the
method of this invention. Examples of vectors for use in this
invention are the AAV-2 based vectors disclosed in Srivastava, PCT
Patent Publication WO 93/09239 or simply a pair of AAV-7 ITRs
having one or more genes operatively positioned therebetween.
[0037] The AAV ITRs employed in the vectors and virions of the
present invention may be the native (wild-type)AAV ITRs or they may
be modified. If the ITRs are modified, they are preferably modified
at their D-sequences. The native D-sequences of the AAV ITRs are
sequences of twenty consecutive nucleotides in each AAV ITR (i.e.,
there is one sequence at each end) which are not involved in HP
formation. The D-sequences of the ITRs are modified by the
substitution of nucleotides, such that 5-18 native nucleotides,
preferably 10-18 native nucleotides, most preferably 10 native
nucleotides, are retained and the remaining nucleotides of the
D-sequence are deleted or replaced with non-native, i.e.,
exogenous, nucleotides. One preferred sequence of five native
nucleotides that are retained is 5' CTCCA 3'. The exogenous or
non-native replacement nucleotide may be any nucleotide other than
the nucleotide found in the native D-sequence at the same position.
For example, appropriate replacement nucleotides for native
D-sequence nucleotide C are A, T and G, and appropriate replacement
nucleotides for native D-sequence nucleotide A are T, G and C. The
construction of four such AAV vectors is disclosed in U.S. Ser. No.
08/921,467, filed Sep. 2, 1997. Other employable exemplary vectors
are pWP-19 and pWN-1, both of which are disclosed in Nahreini, Gene
124 (1993) 257-62. Another example of such an AAV vector is psub201
as disclosed in Samulski, J. Virol. 61 (1987) 3096.
[0038] Other suitable AAV vectors are the Double-D ITR vector.
Methods for making the double-D ITR vectors are disclosed in U.S.
Pat. No. 5,478,745. Still other suitable AAV vectors are those
disclosed in U.S. Pat. No. 4,797,368 (Carter) and U.S. Pat. No.
5,139,941 (Muzyczka), U.S. Pat. No. 5,474,935 (Chartejee) and PCT
Patent Publication WO 94/28157 (Kotin). Yet a further example of an
AAV vector employable in the methods of this invention is
SSV9AFABTKneo, which contains the .alpha.-fetoprotein (AFP)
enhancer and albumin promoter and directs expression of the herpes
simplex thymidine kinase (TK) gene predominantly in the liver. Its
structure and method for making are disclosed in Su, Human Gene
Therapy 7 (1996) 463-70).
[0039] The replication-defective AAV vectors are packaged into
empty AAV capsids to produce the replication-defective AAV virions
helper viruses employed in the methods of the present invention. To
package the replication- defective AAV vectors, which are typically
one or more genes positioned between a pair of ITRs, one employs a
helper construct or helper virus that has AAV-derived coding
sequences that function in trans to enable AAV replication, and
that include the AAV rep and cap sequences. The helper virus has
AAV coding sequences but lacks the AAV ITRs and thus are not
packaged in the capsids that are produced. This helper virus then
provides for transient expression of the AAV rep and cap genes
missing from the AAV vector. For greater details, including
exemplary AAV helper constructs, see, for example, Samulski, J.
Virol 63 (1989) 3822-28; McCarty, J. Virol 65 (1991) 293645 and
U.S. Pat. No. 5,139,941. One such AAV helper construct comprises
pKS rep/cap, which contains the genes encoding the AAV-2 rep and
cap polypeptide sequences. Additional examples of helper viruses,
constructs and functions that can be employed include the plasmids
pAAV/Ad and pIM29+45 (see Samulski, J. Virol. 63 (1989) 3822-28 and
McCarthy, J. Virol 65 (1991) 293645) and those disclosed in U.S.
Pat. No. 5,622,856.
[0040] Accessory functions and accessory function vectors are
non-AAV derived functions and vectors containing sequences encoding
such functions upon which AAV is dependent for its replication.
Such accessory functions can be derived or obtained from any of the
known helper viruses, such as adenovirus, herpesvirus (except
herpes simplex virus type-1) and vaccinia virus and include
moieties and/or sequences involved in activation of gene
transcription, DNA replication, synthesis of cap expression
products and capsid assembly. See, for example, Carter,
"Adeno-Associated Virus Helper Functions" in CRC handbook of
Parvoviruses, Vol. I (1990) (P. Tijssen, ed.); Muzyczka, Current
Topics in Microbiol. 158 (1992) 97-129; Janik, Proc. Natl. Acad Sci
78 (1981) 1925-29; Young, Prog. Med Virol. 25 (1979) 1213 and
Schlehofer, Virology 152 (1986) 110-17.
[0041] The adenoplasmid accessory constructs employed in the method
of the present invention comprise adenovirus plasmid DNA rendered
unable to be packaged into adenovirus particles, for example, the
adenoplasmid accessory constructs lack the adenovirus packaging
signals required for production of infectious adenovirus particles
but contain the adenovirus genes required for rAAV virion
production. Alternatively, the adenoplasmid accessory construct are
rendered to large to be packaged by the additional heterologous
sequences, plasmids or other constructs. Use of such adenoplasmid
accessory constructs results in the generation of rAAV virions
having similar infectious activity and packaging efficiency as
compared to prior art methods.
[0042] One such construct comprises an adenovirus type 5 plasmid
which contains all of the DNA sequence of the serotype 5 adenovirus
but lacks the serotype 5 packaging signal which lies between base
pairs 194 through 398.- See Hearing and Shenk, Cell 33 (1983)
695-703; Hearing, J. Virol. 61 (1987) 2555-58; Grable and Hearing,
J. Virol. 64 (1990) 2047-56; Grable and Hearing, J. Virol. 64
(1990) 723-31. An alternative construct comprises an adenovirus
type 2 plasmid which contains all of the DNA sequence of the
serotype 2 adenovirus but lacks the serotype 2 packaging signal,
which lies in a similar location. analogously, constructs
comprising any other adenovirus serotype may be used, as long as
the packaging signal is removed. Exemplary serotypes which can be
employed include serotypes Ad1, Ad6, Ad8, Ad9, Ad10, Ad11, Ad12,
Ad13, Ad15, Ad17, Ad19, Ad20, Ad22, Ad23, Ad24, Ad25, Ad26, Ad27,
Ad28, Ad29, Ad30, Ad32, Ad33, Ad367, Ad37, Ad38, Ad39, Ad40, Ad41
and Ad42. See Fields Virology, 2 (Fields and Knipe, eds.),
1990.
[0043] Another such construct comprises an adenovirus 5 plasmid
which contains heterologous sequences making it too large to be
packaged. For example, the insertion of plasmid pBR322 or one of
its derivatives at base pair (bp) 1339 (3.7 mu) in the Adenovirus 5
sequence makes the resulting viral genome too large to package. See
Bett, Proc. Natl. Acad. Sci. 91 (1994) 8802-06 and McGrory,
Virology 163 (1988) 614-17. An alternative construct comprises an
adenovirus type 2 plasmid which contains all of the DNA sequence of
the serotype 2 adenovirus but which contains an insertion of pBR322
at a similar location. Analogously, constructs comprising any other
adenovirus serotype may be used, as long as the construct is
rendered too large to be packaged. Exemplary serotypes which can be
employed include Ad1, Ad6, Ad8, Ad9, Ad10, Ad11, Adl2, Adl3, Adl5,
Adl7, Adl9, Ad20, Ad22, Ad23, Ad24, Ad25, Ad26, Ad27, Ad28, Ad29,
Ad30, Ad32, Ad33, Ad367, Ad37, Ad38, Ad39, Ad40, Ad41 and Ad42. See
Fields Virology, 2 (Fields and Knipe, eds.), 1990.
[0044] The adenoplasmid accessory constructs can alternatively
include one or more polynucleotide homologues having substantially
identical functions as the native sequence replacing the native
adenoviral sequences. Such homologues may be derived from a
different adenovirus serotype (since the nucleotide sequence of the
adenovirus type 5 genome is believed to be 99% homologous to the
adenovirus type-2 genome), from another accessory virus or from
another suitable source.
[0045] The adenoplasmid can be in the form of a circularized or
linearlized DNA fragment capable of replication when associated
with appropriate control elements and which can be transcribed and
expressed in a host cell. It can be engineered using conventional
recombinant techniques. For example, the adenoplasmid can be
assembled by inserting adenovirus nucleotide sequences (either
derived from an adenovirus genome, from an adenovirus vector or
chemically synthesized) having accessory functions into a vector
construct in any desired order, for example, by ligating
restriction fragments into the plasmid using polylinker
oligonucleotides. The sequences can then be excised from the vector
and inserted into an appropriate expression plasmid using
techniques well known in the art. One such construct employed in
the examples includes plasmid pBHG10, which is a bacterial plasmid
that contains the Ad5 sequences required to produce infectious
virus upon transfection of 293 cells but lacks the packaging
signal, base pairs 194 through 358 needed to encapsidate viral DNA,
since it contains a deletion of Ad5 sequences from bp 188 through
bp 1339 (0.5 through 3.7 mu). An ampicillin resistance gene and
bacterial origin of replication substitute for the deleted Ads
sequences and the plasmid also lacks the Ads E3 region, from 78.3
through 85.8 map units (mu). Details of its structure and its
construction is described in Bett, Proc. Natl. Acad. Sci. 91 (1994)
8802-06. It is available from Microbix Biosystems, Inc., Ontario,
Canada.
[0046] Other adenoplasmid constructs which can be employed include
plasmid pBG11, which is non-infectious when transfected into 293
cells, since it contains the same deletion of Ads packaging signal
sequences as pBHG10 but lacks the Ads E3 region from 77.5 through
86.2 mu. Details of its structure and its construction is described
in Bett, Proc. Natl. Acad. Sci. 91 (1994) 8802-06.
[0047] Another exemplary adenoplasmid construct contemplated for
use in the invention is pJM17. Construct pjM7 contains an insertion
of a derivative of plasmid pBR322 at base pair (bp) 1339 (3.7 mu)
in its Adenovirus 5 sequence, which makes the resulting viral
genome too large to package. See Bett, Proc. Natl. Acad. Sci. 91
(1994) 8802-06 and McGrory, Virology 163 (1988) 614-17. The
construct pJM17 was derived from pFG140 (see Graham, EMBO J 3
(1984) 2917-22), such that vectors generated with pJM17 also
contain the same deletion(s) and substitution(s) present in the E3
region of its Ad5 sequence as is present in the vector dl 309 (see
Jones, Cell 17 (1979) 683-89).
[0048] The adenoviral gene regions in the adenoplasmid are operably
linked to control sequences that direct their transcription or
expression. Such control sequences can comprise the adenoviral
control sequences associated with the gene regions of the wild-type
adenoviral genes or can comprise heterologous control sequences,
such as heterologous promoters derived from mammalian or viral
genes. Exemplary heterologous promoters include adenoviral
promoters from homologous adenoviruses (i.e., from a different
adenoviral serotype), the SV40 early promoter, the mouse mammary
tumor virus LRT promoter; the adenovirus major late promoter; a
herpes simplex virus promoter, a cytomegalovirus promoter, a rous
sarcoma virus promoter, synthetic promoters or hybrid promoters.
Such promoters are commercially available.
[0049] The adenoplasmid accessory construct can also include one or
more selectable markers. Suitable markers are sequences that confer
antibiotic resistance or sensitivity, impart color, or change the
antigenic character of transfected cells when grown in suitable
selective media. Exemplary selectable markers include the
hygromycin B resistance gene, the ampicillin resistance gene and
the kanamycin resistance gene. Other suitable markers are well
known in the art.
[0050] Alternatively, suitable host cells may provide one or more
of the necessary accessory functions. For example, the human cell
line 293 is a human embryonic kidney cell line that has been
transformed with adenovirus type 5 DNA fragments so that it
expresses adenovirus E1a and E1b genes. Thus, in one embodiment, an
adenoplasmid is provided which lacks the packaging signal and the
E1a and E1b gene regions. Upon transfection into a 293 hot cell,
the adenoplasmid will provide the accessory functions supportive of
rAAV virion production, without the formation of infectious
adenovirus particles.
[0051] The adenoplasmids of the invention can be employed in
methods for the production of rAAV virions. One such method entails
introducing into a suitable host cell an AAV vector, an AAV helper
construct and an adenoplasmid accessory construct into the host
cell. The adenoplasmid accessory plasmid is composed of adenovirus
plasmid DNA lacking packaging signal sequences as described above.
The host cell is cultured to produce crude rAAV virions and lysed.
The resulting cell lysate is applied to a chromatographic column or
a cesium chloride density gradient and the purified rAAV virions
are recovered from the column.
[0052] The heterologous nucleotide sequence(s) that are inserted
into the replication-defective AAV vectors and virions of the
present invention encode one or more therapeutic agents that
include a therapeutic protein, polypeptide, antisense RNA or a
ribozyme, or a combination thereof. Typically, the vectors or
virions contain from one to two therapeutic agents that are native
or non-native to the recipient cell but which have a desired
biological or therapeutic effect.
[0053] As disclosed above, the heterologous nucleotide sequences
that are introduced into the replication-defective AAV vectors and
virions of the present invention include a gene that encodes a
therapeutic protein or polypeptide, preferably a human protein or
polypeptide. Examples of therapeutic proteins and polypeptides that
would be suitable for expression in the methods of the present
invention include the LDL receptor, Factor VIII, Factor IX,
phenylalanine hydroxylase, ornithine transcarbamylase, or
.alpha.1-antitrypsin; a cytokine, such as interleukin (IL)-1, IL-2
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,
IL-13, IL-14 and IL-15, .alpha.-interferon, .beta.-interferon the
.gamma.-interferons, tumor necrosis factor CD3, ICAM-1, LFA-1, or
LFA-3, a chemokine including RANTES 1.alpha., or MIP-1.beta. (see
Cocci, Science 70 (1996) 1811-15); a colony stimulating factor,
such as G-CSF, GM-CSF and M-CSF; growth factors such as IGF-1 and
IGF-2; human hormones such as growth hormone, insulin, calcitonin,
prolactin, follicle stimulating hormone, luteinizing hormone,
chorionic gonadotropin or thyroid stimulating hormone; any one of
the hepatitis genes; thrombopoietin, erythropoietin, or leptin or a
combination of the above. The nucleotide coding sequences for these
proteins and polypeptides are already known in the art. Even more
sequences expressible in the methods and compositions of the
invention include Protein S and Gas6, thrombin, Coagulation Factor
Xa, acidic fibroblast growth factor (FGF-1), basic FGF (FGF-2),
keratinocyte growth factor (KGF), TGF, platelet derived growth
factor (PDGF), epidermal growth factor (EGF), hepatocyte growth
factor (HGF) and HGF activators, PSA, nerve cell growth factor
(NCGF), glial cell derived nerve growth factor (GDNF), vascular
endothelial growth factor (VEGF), Arg-vasopressin, thyroid hormones
asoxymethane, triodothyronine, LIF, amphiregulin, soluble
thrombomodulin, stem cell factor, osteogenic protein 1, the bone
morphogenic proteins, MFG, MGSA, heregulins and melanotropin.
Preferred proteins include but are not limited to erythropoietin,
thrombopoietin (G-CSF), Factor VIII, Factor IX, Factor Xa, human
growth hormone, leptin and IL-2, the DNA sequences of which are all
known in the art, particularly the human DNA sequences.
[0054] The AAV vector may also include control 'sequences, such as
promoters and polyadenylation sites, selectable markers, reporter
genes, enhancers and other control elements permitting for
transcription induction and/or selection. Such AAV vectors can be
constructed using techniques well known in the art.
[0055] The replication-defective AAV helper construct is used to
complement the AAV vector by providing those genes, which are
necessary for the production of AAV virions, particularly the cap
structural genes. Suitable helper constructs having complementing
functions are well known in the art.
[0056] The AAV vector, the replication-defective AAV helper
construct and adenoplasmid accessory construct are introduced into
the host cell either simultaneously or sequentially, using any of
the well known, art recognized transfection techniques, for
example, by calcium phosphate co-precipitation. Culture conditions
include incubating in the range of 35.degree.-40.degree. C. for
approximately 48 to 120 hours. The cells are collected and a lysate
produced using three freeze/thaw cycles and/or sonication. The
lysates are then centrifuged to remove cell debris and the rAAV
virions purified by cesium chloride equilibrium gradient
centrifugation. Any residual adenoviral particles are inactivated
by heating the purified rAAV preparation to at least 56.degree. C.
for 20-30 minutes. Alternatively, the rAAV virions can be purified
by sulfonated cellulose column chromatography following the
protocol described in Tamayose, Human Gene Therapy 7 (1996)
507-513.
[0057] Examples of the above provided description are provided
below.
EXAMPLE 1
Construction of AAV Vector pCMVAAV-LacZ, Helper Plasmid PKSrep/cap
and Adenovirus Plasmids pJM17, pBHG10 and pBHG11
[0058] Vector pKm 201 CMV is a cloning vector in which an
expression cassette containing a CMV immediate early enhancer,
promoter and intron and a bovine growth hormone poly adenylation
site is flanked by AAV-2 ITRs. pKm 201 CMV was derived from pKm20l,
a modified AAV vector plasmid in which the ampicillin resistance
gene of pEMBL-AAV-ITR (Srivastava, (1989)) has been replaced with
the gene for kanamycin resistance. The expression cassette from
pCMVlink, a derivative of pCMV6c (Chapman, (1991), in which the bGH
poly A site has been substituted for the SV40 termination site, was
inserted between the ITRs of pKm201 to generate pKm201CMVLINK. To
construct pCMVAAV-lacZ, the lacZ cDNA sequence was excised from the
plasmid pCMV.beta. (Clontech, Palo Alto, Calif.) and inserted into
pKm201CMVLINK. The plasmid pKm201CMVLINK has the backbone identical
to vector pAAV-TK-MCSFa, which has been deposited with the ATCC, as
Accession No. 98335.
[0059] The AAV helper plasmid, pKSrep/cap was constructed by
cloning the AAV-2 genome without the ITRs, i.e., nucleotides 192
through 4493 of AAV-2 (see Srivastava, J. Virol. 45 (1983) 555-64)
into pBluescript II KS+(Strategene, La Jolla, Calif.).
[0060] Adenovirus plasmids pJM17, pBHG10 and pBHG11 are described
in Bett, Proc. Natl. Acad. Sci. 91 (1994) 8802-06. Plasmid pJM17 is
a non-infectious (replication-defective) adenovirus plasmid when
transfected into human embryonic kidney cells (293 cells), since it
contains an insertion of a derivative of plasmid pBR322 at base
pair (bp) 1339 (3.7 mu) in its Adenovirus-5 sequence, which makes
the resulting viral genome too large to package. Plasmid pBHG10 is
an Adenovirus-5 plasmid that is non-infectious when transfected
into 293 cells, since it contains a deletion of Adenovirus type 5
(AdS) sequences from bp 188 through bp 1339 (0.5 through 3.7 mu),
which removes the packaging signals (psi) required to encapsidate
the adenoviral DNA. An ampicillin resistance gene (Ap) and
bacterial origin of replication (Ori) substitute for the deleted
(bp 188 to 1339) Ad5 sequences. This plasmid also lacks the Ad5 E3
region (from 78.3 through 85.8 mu). Plasmid pBHG11 is an Ad5
plasmid that is infectious when transfected into 293 cells, since
it contains a deletion of Ad5 sequences from bp 188 through bp 1339
(0.5 through 3.7 mu) which removes the packaging signals (psi)
required to encapsidate the adenoviral DNA. An ampicillin
resistance gene (Amp.sup.r) and bacterial origin of replication
(Ori) substitute for the deleted Ad5 sequences. This plasmid also
lacks the Ad5 E3 region (from 77.5 through 86.2 mu).
EXAMPLE 2
Generation of rAAV Particles by Triple Transfection
[0061] For generation of replication defective rAAV particles
(virions) by the triple transfection method of the present
invention, the transient plasmid transfection protocol disclosed in
Zhou (1994) was followed with minor modifications. Human embryonic
kidney cells (293 cells) available from the ATCC under Accession
No. CRL1573 (see also, Graham, J. Gen. Virol. 36 (date) 59-72) were
grown in sterile IMDM medium (Biowhittaker, Mass.) containing 10%
fetal bovine serum at 37.degree. C. in 5% CO.sub.2. Once the cells
had reached 60-70% confluency on a 15 cm dish, the cells were
triple transfected with a mixture comprising the AAV vector, the
replication-defective AAV helper plasmid and the adenoviral plasmid
by the calcium phosphate co-precipitation method. In particular, a
mixture of 10 .mu.g of the AAV vector plasmid, 10 .mu.g of the
replication-defective AAV helper plasmid and 20 .mu.g of the
adenoplasmid pBHG10 was added to 2.5 ml of 250 mM CaCl.sub.2 and
mixed with 2.5 ml of 2.times.HBS (Jordan, Nucleic Acids. Res. 24
(1996) 596-601). The precipitate was left on the cells for eight
hours and replaced with fresh IMDM medium containing 10% fetal
bovine serum (FBS). At 24, 48, 72, 96 and 120 hours after
transfection, the cells were harvested with Hepes buffer (2.5
ml/dish) and lysed by three cycles of freezing and thawing. The
cell lysates were centrifuged at 12,000.times.g for twenty minutes
to remove cell debris. The packaged AAV particles were purified
through two rounds of cesium chloride equilibrium gradient
centrifugation and residual adenoviral particles were inactivated
by heat treatment at 56.degree. C. for thirty minutes.
Alternatively, the packaged AAV particles are purified by
sulfonated cellulose column chromatography as described in
Tamayose, Human Gene 7therapy 7 (1996) 507-13.
[0062] Another set of transfections involved an identical protocol,
except that the adenoplasmid DNA was not used; instead, eight hours
post transfection the cells were infected with adenovirus dl 312 at
a multiplicity of infection (MOI) of 2. Adenovirus dl 312 has a
deletion in the E1a gene and is propagated in 293 cells transformed
with left-end of adenovirus sequences. It expresses E1a and E1b
transcripts. See Moran Cell 48 (1987) 177-78. The transformed cells
were harvested at 72 hours post-infection and purified as described
above.
[0063] For estimation of the total number of vector particles
produced, the purified vector stock was treated with DNAse I and
the encapsidated DNA was extracted with phenol-chloroform,
precipitated with ethanol, and subject to dot blot analysis as
described in Nahreini and Srivastava, Interviriology 30 (1989)
74-85. 293 cells were then infected with serial dilutions of vector
stock. The positive cells were counted and the functional titer was
estimated. For wild-type AAV testing, the rAAV stock was diluted
serially and 293 cells (2.times.10.sup.5) were infected along with
Ad at an MOI of 2. Three days later the cells were harvested, lysed
by three freeze/thaw cycles and the cell debris removed by
centrifugation. The supernatant was heat-inactivated for ten
minutes and fresh 293 cells (6.times.10.sup.6) were infected in the
presence of Ad at an MOI of 2. At 48 hours post-infection, the low
molecular weight DNA was isolated following the method of Hirt, J.
Mol. Biol. 26 (1967) 365-69, fractionated on an agarose gel and
transferred to a nylon membrane. The blot was hybridyzed to a
biotinylated oligonucleotide probe specific for the AAV capsid
region. The titer was reported base on the highest dilution of the
vector stock showing positive signal for AAV capsid DNA.
[0064] To visualize the amount of adeno- and AAV protein in the
preparations by Western blot analysis, aliquots from the crude
lysate and final preparation (5 .mu.l (0.008% of the total) of the
freeze/thaw lysate and 5 .mu.l (0.5% of the total) of the final
products) were electrophoresed on 10% Tris-Glycine gels (Novex, San
Diego, Calif.) and electroblotted onto nitrocellulose. The blots
were probed with either a monoclonal antibody against AAV capsid
protein (ARP Inc., Belmont, Mass.) and a goat anti-mouse (IgG HRP
conjugate (Bo-Rad Laboratories, Richmond, Calif.) or a polyclonal
antibody against adenovirus type 5 (DAKO, Denmark) and a goat
anti-rabbit IgG HRP conjugate. The signal was detected using a
chemiluminescence detection kit (ECTL, Amersham, Bukinghamshire,
Great Britain).
[0065] The results of the Western blot analysis of adenovirus
proteins are as follows. Lane 1, which contained the crude
preparation (0.008% of the total) from the standard protocol,
showed an extensive smear of protein extending from about 29 kD to
over 140 kD. Under similar testing conditions, lane 2, which
contained the crude preparation (0.008%) from the triple
transfection protocol, showed very low levels of adenoproteins. The
purified AAV products, when analyzed for adenovirus protein
contamination, showed still some adenoprotein contamination in
standard protocol (lane 3), whereas the triple transfection
protocol of the present invention had no detectable adenoprotein
contamination (lane 4). For adenovirus contamination testing, the
samples were diluted and added to 50% confluent 293 cells (plated
on 12 well dishes with 1.times.10.sup.5 cells). The cultures were
passaged for at least three weeks or until the cultures exhibited
cytopathic effect. The Control included 293 cells infected with
known amount of adenovirus stock. The limit of detection in the
assay was 100 pfu/ml. These results demonstrated that the triple
transfection protocol does not lead to adenovirus
contamination.
[0066] To determine whether using a replication-defective
adenoplasmid accessory construct in place of a
replication-competent (helper) adenovirus inhibited AAV virion
production, we compared the number of "total" and "functional" rAAV
virions obtained from freeze/thaw lysates/10 cm culture dish for
the standard protocol of the prior art and the triple transfection
protocol of the present invention. Total particles were determined
by dot blot analysis. Functional particles were determined by
staining for X-gal activity and counting positive cells.
1TABLE 1 Comparison of Time Course of rAAV Production By Standard
Versus Triple Transfection Protocols Total Particles Functional
Particles Standard Protocol 72 hours 4.5 .times. 10.sup.11 1
.times. 10.sup.9 Triple Transfection Protocol 24 hours 1.6 .times.
10.sup.11 3.2 .times. 10.sup.7 48 hours 6 .times. 10.sup.11 6.6
.times. 10.sup.8 72 hours 7 .times. 10.sup.11 7.4 .times. 10.sup.8
96 hours 7 .times. 10.sup.11 1.5 .times. 10.sup.8 120 hours 9
.times. 10.sup.11 8.6 .times. 10.sup.7
[0067] The data from Table 1 demonstrate that even in the absence
of a productive (replication-competent) adenovirus infection, the
encapsidation of rAAV particles is efficiently obtained in human
293 cells provided with adenoviral DNA from an adenoplasmid
accessory construct. This experiment also demonstrates that cells
transformed with adenoplasmid accessory element had similar numbers
of total viral particles between 72-120 hours, and that at 72
hours, the total numbers of particles generated by both methods was
comparable and the cell numbers per dish were similar.
EXAMPLE 3
Scale Up of The Triple Transfection Method
[0068] Large-scale transfections were performed as described in
Example 2. Replication-defective AAV particles (virions) encoding a
gene of interest but free of coding sequences for AAV proteins,
were recovered from twenty 15 cm dishes and purified on a CsC1
gradient. Total particles and functional particles were estimated
as described in Example 2. The results are shown below.
2TABLE 2 Comparison of Purified rAAV Particles Generated By
Standard Versus Triple Transfection Protocol Total Particles
Functional Particles Method Lysate Purified Stock Lysate Purified
Stock Standard 6 .times. 10.sup.13 1.5 .times. 10.sup.13 6.4
.times. 10.sup.10 3.9 .times. 10.sup.10 Triple 5.1 .times.
10.sup.13 1.3 .times. 10.sup.13 7.6 .times. 10.sup.10 3.4 .times.
10.sup.10 Transfection
[0069] After CsC1 gradient purification, adenoviral protein
contamination was undetectable by Western blot technique in rAAV
preparations prepared by the triple transfection protocol.
[0070] All patents, patent publications, patent applications and
scientific articles mentioned in this specification are herein
incorporated by reference. The invention now being fully described,
it will be apparent to one of ordinary skill in the art that many
changes and modifications can be make thereto without departing
from the spirit or scope of the appended claims.
* * * * *