U.S. patent application number 14/216778 was filed with the patent office on 2014-10-30 for scalable manufacturing process to produce recombinant lentiviral vectors in serum-free suspension cell culture system.
The applicant listed for this patent is THE CHILDREN'S HOSPITAL OF PHILADELPHIA. Invention is credited to Guang QU, John Fraser WRIGHT.
Application Number | 20140323556 14/216778 |
Document ID | / |
Family ID | 51538029 |
Filed Date | 2014-10-30 |
United States Patent
Application |
20140323556 |
Kind Code |
A1 |
QU; Guang ; et al. |
October 30, 2014 |
SCALABLE MANUFACTURING PROCESS TO PRODUCE RECOMBINANT LENTIVIRAL
VECTORS IN SERUM-FREE SUSPENSION CELL CULTURE SYSTEM
Abstract
Methods for preparing highly purified rLV vector formulations at
the scale needed to meet anticipated demand for human gene therapy
are provided.
Inventors: |
QU; Guang; (Philadelphia,
PA) ; WRIGHT; John Fraser; (Philadelphia,
PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE CHILDREN'S HOSPITAL OF PHILADELPHIA |
Philadelphia |
PA |
US |
|
|
Family ID: |
51538029 |
Appl. No.: |
14/216778 |
Filed: |
March 17, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61787818 |
Mar 15, 2013 |
|
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|
Current U.S.
Class: |
514/44R ;
435/320.1 |
Current CPC
Class: |
C12N 7/00 20130101; C12N
2740/15051 20130101; C12N 2740/15043 20130101; A61K 48/0091
20130101 |
Class at
Publication: |
514/44.R ;
435/320.1 |
International
Class: |
A61K 48/00 20060101
A61K048/00 |
Claims
1. A method for large scale viral vector purification, comprising;
a) harvesting recombinant viral vectors comprising a transgene from
serum free suspension culture; b) clarifying the harvest of step a)
via filtration; c) subjecting the clarified suspension of step b)
to tangential flow filtration to reduce volume and exchange buffer;
d) harvesting the filtrate from step c) and exposing said filtrate
to benzonase digestion to remove DNA/RNA impurities; e) subjecting
the the filtrate of step d) to PEG-modulated weak anion exchange
column chromatography, thereby isolating said viral vectors; f)
further purifying the viral vectors obtained from step e) via
tangential flow filtration to reduce the volume and buffer
exchange; g) subjecting the filtrate of step f) to size exclusion
column chromatography to further purify said viral vectors; h)
subjecting the vectors of step g) to tangential flow filtration,
and thereby obtaining final vector titer; and i) filtering a vector
solution obtained from step h) through a 0.45 um filter; and j)
storing said purified viral vectors.
2. The method of claim 1, wherein said vector is a recombinant
lentiviral vector (rLV) and said rLV is produced at approximately
3.times.10.sup.8/ml.
3. A rLV vector formulation comprising rLV particles purified using
the method of claim 1 in a pharmaceutically acceptable carrier.
4. The method of claim 1, wherein said transgene encodes a nucleic
acid selected from the group consisting of a siRNA, an antisense
molecule, and a miRNA a ribozyme and a shRNA.
5. The method of claim 1 wherein said transgene encodes a gene
product selected from the group consisting of insulin, glucagon,
growth hormone (GH), parathyroid hormone (PTH), growth hormone
releasing factor (GRF), follicle stimulating hormone (FSH),
luteinizing hormone (LH), human chorionic gonadotropin (hCG),
vascular endothelial growth factor (VEGF), angiopoietins,
angiostatin, granulocyte colony stimulating factor (GCSF),
erythropoietin (EPO), connective tissue growth factor (CTGF), basic
fibroblast growth factor (bFGF), acidic fibroblast growth factor
(aFGF), epidermal growth factor (EGF), transforming growth factor
.alpha. (TGF.alpha.), platelet-derived growth factor (PDGF),
insulin growth factors I and II (IGF-I and IGF-II), TGF.beta.,
activins, inhibins, bone morphogenic protein (BMP), nerve growth
factor (NGF), brain-derived neurotrophic factor (BDNF),
neurotrophins NT-3 and NT4/5, ciliary neurotrophic factor (CNTF),
glial cell line derived neurotrophic factor (GDNF), neurturin,
agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF),
ephrins, noggin, sonic hedgehog and tyrosine hydroxylase.
6. The method of claim 1, wherein said transgene encodes a gene
product selected from the group consisting of thrombopoietin (TPO),
interleukins (IL1 through IL-17), monocyte chemoattractant protein,
leukemia inhibitory factor, granulocyte-macrophage colony
stimulating factor, Fas ligand, tumor necrosis factors .alpha. and
.beta., interferons .alpha., .beta., and .gamma., stem cell factor,
flk-2/flt3 ligand, IgG, IgM, IgA, IgD and IgE, chimeric
immunoglobulins, humanized antibodies, single chain antibodies, T
cell receptors, chimeric T cell receptors, single chain T cell
receptors, class I and class II MHC molecules,
7. The method of claim 1, wherein said transgene comprises a
nucleic acid encoding a protein useful for correction of in born
errors of metabolism selected from the group consisting of
carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate
synthetase, arginosuccinate lyase, arginase, fumarylacetacetate
hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin,
glucose-6-phosphatase, porphobilinogen deaminase, factor V, factor
VIII, factor IX, cystathione beta-synthase, branched chain ketoacid
decarboxylase, albumin, isovaleryl-coA dehydrogenase, propionyl CoA
carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase,
insulin, beta-glucosidase, pyruvate carboxylate, hepatic
phosphorylase, phosphorylase kinase, glycine decarboxylase, RPE65,
H-protein, T-protein, a cystic fibrosis transmembrane regulator
(CFTR) sequence, and a dystrophin cDNA sequence.
8. The method of claim 7, wherein the gene product is Factor VIII
or Factor IX.
Description
RELATED APPLICATION INFORMATION
[0001] This application claims priority to application Ser. No.
61/787,818, filed Mar. 15, 2013, which application is expressly
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to the fields of molecular biology
and gene therapy. More specifically, the invention provides
improved processes for large scale production of viral vectors,
preferably lentiviral and adeno-associated viral vectors,
comprising transgenes which encode medically beneficial products
for clinical use.
INTRODUCTION
[0003] Several publications and patent documents are cited
throughout the specification in order to describe the state of the
art to which this invention pertains. Each of these citations is
incorporated herein by reference as though set forth in full.
[0004] Recombinant Lentivirus-based (rLenti) vectors have been
developed and widely used as investigational gene delivery products
for several serious human diseases. Lentiviral vectors have proven
to be very productive in terms of transduction due to their ability
to infect both replicating and non-replicating cells, including
stem cells.
[0005] Many clinical trials have been initiated world-wide using
HIV-1 based, VSVG pseudo-typed lentiviral vectors and very
promising clinical benefits have been observed. However, a
significant problem in the field at this time is a lack of
methodology to make sufficient quantities of rLenti that will be
need for advanced clinical studies. The research and clinical trial
data have shown that rLentivector is a promising gene delivery
vehicle for human gene therapy, for genetic diseases such as
primary immunodeficiencies (Fischer and colleauges) as well as
immunotherapeutics for cancers (June and colleagues).
[0006] However, there is a critical need in the field to develop
scalable production and purification methods which are suitable for
cGMP manufacture of large quantities of rLenti vectors which meet
manufacturing capacity and investigational product quality
requirements to support late stages of clinical applications. For
example, for one very promising programs in Phase I for treatment
of leukemias, it is anticipated that at least 100-fold greater
manufacturing capacity relative the currently available methods
will be required for Phase III studies and early stage licensed
product launch. Thus, methods scaling up production of this gene
therapy vector are urgently needed.
SUMMARY
[0007] In accordance with the invention, methods are provided for
production of high titer rLenti vectors in a scalable, serum-free
suspension cell culture and purification of the vector using
scalable, industry standard column chromatography techniques. An
rLV vector formulation comprising rLV particles produced according
to the methods can be made, and optionally included in a
pharmaceutically acceptable carrier.
[0008] In one embodiment, a method for viral vector purification
includes: a) harvesting recombinant viral vectors comprising a
transgene from serum free suspension culture; clarifying the
harvest of step a) via filtration; c) harvesting the filtrate from
step b) and optionally exposing said filtrate to nuclease digestion
to remove DNA/RNA impurities; subjecting the filtrate of step c) to
PEG-modulated affinity or ion exchange column chromatography,
thereby isolating said viral vectors; further purifying the viral
vectors obtained from step d) via tangential flow filtration to
reduce the volume and buffer exchange; subjecting the filtrate of
step e) to size exclusion column chromatography to further purify
said viral vectors; subjecting the vectors of step f) to tangential
flow filtration, and thereby obtaining final vector titer;
filtering a vector solution obtained from step g) through a filter;
and collecting said purified viral vectors.
[0009] In another embodiment, a method for viral vector
purification includes: a) harvesting recombinant viral vectors
comprising a transgene from serum free suspension culture; b)
clarifying the harvest of step a) via filtration; c) subjecting the
clarified suspension of step b) to tangential flow filtration to
reduce volume and exchange buffer; d) harvesting the filtrate from
step c) and optionally exposing said filtrate to nuclease digestion
to remove DNA/RNA impurities; e) subjecting the filtrate of step d)
to PEG-modulated affinity or ion exchange column chromatography,
thereby isolating said viral vectors; f) subjecting the viral
vectors obtained from step e) to size exclusion column
chromatography to further purify said viral vectors; g) subjecting
the vectors of step f) to tangential flow filtration, and thereby
obtaining final vector titer; h) filtering a vector solution
obtained from step g) through a filter; and i) collecting said
purified viral vectors.
[0010] In a further embodiment, a method for viral vector
purification includes: a) harvesting recombinant viral vectors
comprising a transgene from serum free suspension culture; b)
clarifying the harvest of step a) via filtration; c) subjecting the
clarified suspension of step b) to tangential flow filtration to
reduce volume and exchange buffer; d) harvesting the filtrate from
step c) and optionally exposing said filtrate to nuclease digestion
to remove DNA/RNA impurities; e) subjecting the filtrate of step d)
to PEG-modulated affinity or ion exchange column chromatography,
thereby isolating said viral vectors; f) further purifying the
viral vectors obtained from step e) via tangential flow filtration
to reduce the volume and buffer exchange; g) subjecting the
filtrate of step f) to size exclusion column chromatography to
further purify said viral vectors; h) subjecting the vectors of
step g) to tangential flow filtration, and thereby obtaining final
vector titer; filtering a vector solution obtained from step h)
through a filter; and j) collecting said purified viral
vectors.
[0011] Methods of the invention are applicable to lentiviral
vectors (rLV). In particular embodiments, an rLV vector comprises a
recombinant lentiviral vector (rLV) is an HIV-1, HIV-2, HIV-1/HIV-2
pseudotype, HIV-1/SIV, FIV, caprine arthritis encephalistis virus
(CAEV), equine infectious anemia virus, bovine immunodeficiency
virus, HIV and their pseudotypes, or a Vesucular Stomatitis Virus
G-pseudotyped lentivirus (VSVG pseudotypede) vector.
[0012] Viral vectors in accordance with the invention include
transgenes. In particular embodiments, a transgene encodes a
nucleic acid selected from the group consisting of a siRNA, an
antisense molecule, and a miRNA a ribozyme and a shRNA. In
additional particular embodiments, a transgene encodes a gene
product (protein or polypeptide).
[0013] In particular aspects, a gene product (protein or
polypeptide) is insulin, glucagon, growth hormone (GH), parathyroid
hormone (PTH), growth hormone releasing factor (GRF), follicle
stimulating hormone (FSH), luteinizing hormone (LH), human
chorionic gonadotropin (hCG), vascular endothelial growth factor
(VEGF), angiopoietins, angiostatin, granulocyte colony stimulating
factor (GCSF), erythropoietin (EPO), connective tissue growth
factor (CTGF), basic fibroblast growth factor (bFGF), acidic
fibroblast growth factor (aFGF), epidermal growth factor (EGF),
transforming growth factor .alpha. (TGF.alpha.), platelet-derived
growth factor (PDGF), insulin growth factors I and II (IGF-I and
IGF-II), TGF.beta., activins, inhibins, bone morphogenic protein
(BMP), nerve growth factor (NGF), brain-derived neurotrophic factor
(BDNF), neurotrophins NT-3 and NT4/5, ciliary neurotrophic factor
(CNTF), glial cell line derived neurotrophic factor (GDNF),
neurturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor
(HGF), ephrins, noggin, sonic hedgehog or tyrosine hydroxylase. In
additional particular aspects, a gene product (protein or
polypeptide) is thrombopoietin (TPO), interleukins (IL1 through
IL-17), monocyte chemoattractant protein, leukemia inhibitory
factor, granulocyte-macrophage colony stimulating factor, Fas
ligand, tumor necrosis factors .alpha. and .beta., interferons
.alpha., .beta., and .gamma., stem cell factor, flk-2/flt3 ligand,
IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized
antibodies, single chain antibodies, T cell receptors, chimeric T
cell receptors, single chain T cell receptors, G protein-coupled
receptors (GPCRs), CCR5, and class I and class II MHC
molecules.
[0014] In further particular aspects, a gene product (protein or
polypeptide) is a nucleic acid encoding a protein useful for
correction of in born errors of metabolism selected from the group
consisting of carbamoyl synthetase I, ornithine transcarbamylase,
arginosuccinate synthetase, arginosuccinate lyase, arginase,
fumarylacetacetate hydrolase, phenylalanine hydroxylase, alpha-1
antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase,
factor V, factor VIII, factor IX, cystathione beta-synthase,
branched chain ketoacid decarboxylase, albumin, isovaleryl-coA
dehydrogenase, propionyl CoA carboxylase, methyl malonyl CoA
mutase, glutaryl CoA dehydrogenase, insulin, beta-glucosidase,
pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase,
glycine decarboxylase, retinal pigment epithelium-specific 65 kDa
protein (RPE65), H-protein, T-protein, a cystic fibrosis
transmembrane regulator (CFTR) sequence, or a dystrophin cDNA
sequence. In still additional particular aspects, a gene product
(protein or polypeptide) is Factor VIII or Factor IX.
[0015] In still further particular aspects, a transgene encodes a
tumor associated antigen (TAA). In yet further particular aspects,
a transgene encodes a gene product of any of CAIX, CD19, CD20,
CD20, CD22, CD30, CD33, CD44v7/8, CEA, EGF-RIII (epidermal growth
factor receptor variant 3) EGP-2, erb-B2, erb-B2, 3, 4, FBP, fetal
acetycholine receptor, GD2, Her2/neu, IL-13R-a2, KDR, k-light
chain, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, MUC1,
NKG2D, oncofetal antigen (h5T4), PSCA, PSMA, mAb IgE targeted TAA,
TAG-72 or VEGF-R2.
[0016] Viral vectors in accordance with the invention may be
produced by cells. In particular embodiments, recombinant viral
vectors are produced by mammalian cells. In particular aspects,
recombinant viral vectors are produced by HEK 293T (ATCC); HEK293F
(Life Technologies); HEK293(ATCC); 293S (ATCC), BHK (ATCC), BHK-21
(ATCC), CHO (ATCC), CHO/dhFr- (ATCC)1, or CHO K1 (ATCC) cells.
[0017] Cells producing recombinant viral vectors in accordance with
the invention are typically grown in suspension in a growth medium.
Growth medium for cells include serum free cell growth medium. In
particular aspects, a serum free growth medium is FreeStyle.TM.293
(Gibco.RTM., Life Technologies), DMEM/F12 (Gibco.RTM., Life
Technologies), SFM4Transfx-293 (HyClone.TM., ThermoScientific),
CDM4HEK293 (HyClone.TM., ThermoScientific), StemPro-34SFM
(Gibco.RTM., Life Technologies), FreeStyle F17 (Gibco.RTM., Life
Technologies), 293SFM II (Gibco.RTM., Life Technologies), or CD293
(Gibco.RTM., Life Technologies), or a combination thereof.
[0018] Nucleases can be employed in the invention methods. In
particular embodiments, a nuclease is an endonuclease, an
exonuclease, or a combination thereof. In additional particular
embodiments, a nuclease is a deoxyribonuclease, a ribonuclease, or
a combination thereof. In further particular embodiments, a
nuclease is a benzonase or a DNase.
[0019] Various resins or chromatography substrates (media) can also
be employed in the invention methods. In particular embodiments,
affinity or ion exchange resin or substrate (media) can be
employed. In particular aspects, ion exchange column chromatography
comprises anion or cation exchange column chromatography, strong or
weak anion exchange, or strong or weak cation exchange.
[0020] Methods of the invention also include additional steps of
binding, washing and/or eluting in connection with column
chromatography. Such binding, washing and/or eluting steps can be
performed one or multiple times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,
10 times). In various embodiments, a method includes adjusting the
filtrate of a prior step to be a binding solution (for binding of
virus to the resin or media of the column); and/or contacting the
filtrate with the affinity or ion exchange column thereby binding
the viral vectors to the affinity or ion exchange column, and/or
washing the bound viral vectors to remove impurities with a washing
solution, the solution optionally including PEG or PEG and a salt;
and/or eluting the viral vectors from the affinity or ion exchange
column with an elution solution.
[0021] In particular aspects, a binding solution includes PEG in an
amount from about 0% to 10% weight/volume, or from about 0% to 5%
weight/volume, or from about 0% to 2% weight/volume. In additional
particular aspects, a binding solution includes PEG having a
molecular weight from about 2,000 kDa to about 40,000 kDa.
[0022] In particular aspects, a washing solution includes PEG in an
amount from about 1% to 10% weight/volume, or from about 1% to 5%
weight/volume, or from about 1% to 2% weight/volume. In additional
particular aspects, a washing solution includes PEG having a
molecular weight from about 2,000 kDa to about 40,000 kDa.
[0023] In particular aspects, an elution solution includes PEG in
an amount from about 0% to 20% weight/volume. In additional
particular aspects, an elution solution includes PEG having a
molecular weight from about 2,000 kDa to about 40,000 kDa.
[0024] In invention methods, binding, washing and/or elution
solutions can optionally include one or more salts. In particular
embodiments, binding, washing and/or elution solution includes one
or more salts in an amount from about 20 mM to about 1,000 mM (1M).
Non-limiting salts include sodium chloride and/or potassium
chloride.
[0025] In additional embodiments, a method for viral vector
purification includes one or more filtering steps. In particular
embodiments, filtering is through a filter having a pore diameter
of about 0.20-0.5 um. In particular aspects, filtering is through a
0.20 um pore diameter filter, a 0.22 um pore diameter filter, or a
0.45 um pore diameter filter.
[0026] As disclosed herein, methods of the invention are able to
produce high titers of purified viral vectors, for example, from
1.times.10.sup.5 infectious units (IU)/ml viral vector, up to
approximately 1.times.10.sup.9 infectious units (IU)/ml viral
vector. In particular embodiments, viral vector is produced at
approximately 5.times.10.sup.5 infectious units (IU)/ml, viral
vector is produced at approximately 6.times.10.sup.6 infectious
units (IU)/ml, or viral vector is produced at approximately
3.times.10.sup.8 infectious units (IU)/ml.
DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1A-1D. Flow charts of exemplary methods for
purification of lentiviral vectors of the invention.
[0028] FIG. 2A-2C. Characterization of adapted HEK 293T cell growth
in serum free suspension culture. A: Light microscope image
depicting cell population adapted in CD293 serum free media, no
cell aggregation observed. B. Optimization of cell culture
conditions using spinner flasks. Under the condition of 8% CO2 and
130 rotation per min, the cells can be cultured for four days and
about 3E+06 cells per ml. C. Cell growth rate is consistent with
doubling time about 20 hours.
[0029] FIG. 3A-3D. Fluorescent microscope images depicting eGFP
expression in PEI transfected HEK 293 T cells in serum free
suspension culture. Panel A, B, C and D are cells in different
serum free culture media. eGFP positive cells are detected only in
Panel D.
[0030] FIG. 4. Fluorescent microscope images depicting eGFP
expression in PEI transfected HEK 293 T cells in serum free
suspension culture. Panel A transfected with PEI 25,000 kDa; B
transfected with PEI MAX (40,000 kDa); Panel C transfected with PEI
pro. PEI Max resulted highest transfection efficiency.
[0031] FIG. 5. Fluorescent microscope images depicting PEI
transfection efficiency of HEK 293 T in serum free suspension
culture (A) and lentiviral vector tranduction of HEK 293 cells.
Panel A transfected using optimized condition with PEI MAX (40,000
kDa); Panel B shows lentivector transduction (50 ul harvested
supernatant used).
[0032] FIG. 6A-6E. Analysis of Lentivector recovery from DEAE
column chromatography and PEG modulated DEAE column Chromatography.
Top panel illustrates the eGFP expression of samples from column
chromatography fractions. A: Chromatography does not contains PEG
modulation; B, C and D contains additional washing step using 4%
PEG (4K); 4% PEG (6K) and 8% PEG (4K). 100 ul of each fraction were
used to transduce HEK293 cells, eGFP positive cells were detected
using fluorescence microscope and FACS (Bottom Panel). Almost 100%
Lentivectors were detected in the elution fractions.
[0033] FIG. 7A-7D. Analysis of Lentivector elution using UV 280 nm
absorption. 30 mls clarified lentivectors harvest were loaded on a
16 ml DEAE Sepharsoe FF column. Lentivectors were purified with PEG
modulation (Panel B, C, D) or without PEG modulation (A). The
vector elution peak was integrated using UV 280 nm absorption, the
peak area reduce from 5 fold to almost 20 fold. It is interesting
to note that UV absorption ratio of UV280/UV260 changed from UV280
dominant (panel A) to UV 260 dominant (panel B, C and D),
indicating improved vector purity.
[0034] FIG. 8. SDS-PAGE Analysis of the Purity of Lentivector
Eluted from Column Chromatography. 10 .mu.l of each samples were
loaded on a 4-12% NuePage Bis-Tris gel and silver stained after
electrophoresis. Elution A is the sample of lentivector elution
from column chromatography without PEG modulation; Elution B, C and
D are the elution fractions from PEG-modulated column
chromatography using 4% 4K PEG; 4% 6K PEG and 8% 4K PEG
respectively. While the Lentivector recoveries are highly
comparable for all of these eluted samples, the protein impurities
are significantly reduced in the PEG-modulated fractions.
[0035] FIG. 9. FACS analysis of rLentivector productivity.
Recombinant Lentivector expressing eGFP were produced using an
optimized transfection method in serum-free suspension cell culture
from 12 well-plates (1.5 ml cell culture) or spinner flask (40 ml
cell culture). Vector yields were determined using FACS analysis.
Vector productivity up to 6E+06 transduction units (TU) per
milliliter were observed from both 12 well plates and spinner cell
culture.
DETAILED DESCRIPTION
[0036] Recombinant Lentivirus vectors are typically produced by
using transfection methods in research. There are several methods
that are known in the art for generating rLV virions: for example,
transfection using vector and rLV helper sequences (see for
example, Merten et al 2011). However, when manufacturing rLenti
vectors for clinical application, particularly for late stages of
clinical applications, it is highly preferable to manufacture the
vectors using a serum free suspension production system which can
be scaled up to ensure the adequate manufacturing capacity and
ensuring a superior safety profile of the vector manufactured.
[0037] Disclosed herein is a transfection-based production method
to produce high titer of lentiviral vectors in a scalable serum
free cell culture system. Cells from a commercial source, available
as adapted for adherent growth, were successfully adapted into
serum free cell culture media. We evaluated more than five
different cell culture media which are claimed to support
serum-free suspension cell culture, but identified only one media
that support non-aggregated, healthy cell growth after adaption
with fast growth rate. The adapted cells have been cultured in the
serum free suspension culture condition for several months and a
research cell bank has been developed.
[0038] Lentiviruses are enveloped viruses, and are significantly
different in terms of virus structure and life cycle from other
viruses used for delivery of nucleic acid into cells, such as
adeno-associated viruses (AAV). Lentiviruses are composed of 2
copies of RNA, a nuclear capsid (NC), a Capsid (CA) a membrane
associated matrix (MA), envelope proteins such as surface
glycoproteins and transmembrane proteins and enzymes such as
integrase (IN), protease (PR), reverse transcriptase (RT) and
accessory proteins (e.g., Nef, Vif, Vpu, Vpr). Lentiviruses infect
cells by binding of a surface glycoprotein of the virus to a
receptor on the cell. The membranes of the envelope of the virus
and the cell then fuse allowing the virus to enter the cell.
Following entry, uncoating of viral RNA and reverse transcription
takes place which leads to the formation of a pre-integration
complex, which contains double stranded DNA, RT, IN, Vpr (or Vpx in
HIV-2) NC, and some copies of the MA (Suzuki and Craigie 2007,
Depienne et al., 2000, Bukrinsky et al., 1993 and Miller et al.,
1997). Once the provirus enters the nuclear envelope, the viral DNA
integrates within the cell genome. Normal cellular functions of
transcription and translation are followed by assembly of
structural viral proteins with viral RNA and subsequent viral
budding.
[0039] Lentiviruses are desirable for delivery of nucleic acid into
cells in part because they can infect non-dividing cells by
actively entering the nucleus through the nuclear envelope. By
contrast, other retroviruses require cell division for infection
due to the fact that it cannot enter the nuclear envelope of a
non-dividing cell.
[0040] "Lentiviruses" include members of the bovine lentivirus
group, equine lentivirus group, feline lentivirus group,
ovinecaprine lentivirus group and primate lentivirus group.
Examples of lentiviruses suitable for the methods and use of the
invention include, but are not limited to, HIV and their
pseudotypes such as HIV-1, HIV-2, HIV-1/HIV-2 pseudotype,
HIV-1/SIV, FIV, caprine arthritis encephalitis virus (CAEV), equine
infectious anemia virus, bovine immunodeficiency virus and
Vesucular Stomatitis Virus G-pseudotyped lentivirus (VSVG
pseudotypede).
[0041] The development of lentiviral vectors for gene therapy has
been reviewed in Klimatcheva et al., 1999, Frontiers in Bioscience
4: 481-496. The design and use of lentiviral vectors suitable for
gene therapy is described, for example, in U.S. Pat. No. 6,207,455,
issued Mar. 27, 2001, and U.S. Pat. No. 6,165,782, issued Dec. 26,
2000. Additional systems are disclosed in Merten et al. (2011).
[0042] The terms "gag polyprotein", "pol polyprotein", and "env
polyprotein" refer to the multiple proteins encoded by retroviral
gag, pol and env genes which are typically expressed as a single
precursor "polyprotein". For example, HIV gag encodes, among other
proteins, p17, p24, p9 and p6. HIV pol encodes, among other
proteins, protease (PR), reverse transcriptase (RT) and integrase
(IN). HIV env encodes, among other proteins, Vpu, gp120 and gp41.
As used herein, the term "polyprotein" shall include all or any
portion of gag, pol and env polyproteins.
[0043] The terms "Vpx" and "Vpr" refer respectively to lentiviral
Vpx and Vpr proteins described, for example, in WO 96/07741, hereby
incorporated by reference in its entirety. These terms also refer
to fragments, mutants, homologs and variants of Vpr and Vpx which
retain the ability to associate with p6.
[0044] The term "fusion protein" refers to a molecule comprising
two or more proteins linked together. Typically, the fusion protein
is an amino acid sequence comprising two or more protein
sequences.
[0045] By "vector" is meant a genetic element, such as a plasmid,
phage, transposon, cosmid, chromosome, virus, virion, etc., which
is capable of replication when associated with the proper control
elements and which can transfer gene sequences between cells. Thus,
the term includes cloning and expression vehicles, as well as viral
vectors.
[0046] As used herein, the term "recombinant," as a modifier of
sequences such as vector as well as a modifier of a virus, means
that the compositions have been manipulated (i.e., engineered) in a
fashion that generally does not occur in nature.
[0047] A "recombinant lentiviral vector" or "rLV" is a genetic
element comprising a lentivirus linear, double-stranded nucleic
acid genome. The lentivirus linear, double-stranded nucleic acid
genome has been genetically altered, e.g., by the addition or
insertion of a heterologous nucleic acid construct.
[0048] By "recombinant virus" is meant a virus that has been
genetically altered, e.g., by the addition or insertion of a
heterologous nucleic acid construct into the particle. A
recombinant virus does not include infectious virus as they exist
in nature.
[0049] A "LV virion" is meant a complete virus particle, such as a
wild-type (wt) LV virus particle associated with an rLV envelope.
By "rLV virion" is meant a complete virus particle, such as a rLV
virus particle comprising a linear, double-stranded LV nucleic acid
genome and a heterologous nucleotide sequence of interest
associated with an rLV envelope. Examples of rLV suitable for the
methods and uses of the invention include, but are not limited to,
HIV and their pseudotypes such as HIV-1, HIV-2, HIV-1/HIV-2
pseudotype, HIV-1/SIV, FIV, caprine arthritis encephalitis virus
(CAEV), equine infectious anemia virus, bovine immunodeficiency
virus and Vesucular Stomatitis Virus G-pseudotyped lentivirus (VSVG
pseudotyped).
[0050] The terms "recombinant rLV virion," "rLV vector particle,"
and "full particles" are defined herein as an infectious,
replication-defective virus including an rLV membrane envelope, and
a transgene comprising a heterologous nucleotide sequence of
interest. A review describing rLV molecular features is provided in
Dropulic (2011). As set forth herein, a "recombinant rLV virion"
does not include infectious LV as they exist in nature.
[0051] The term "host cell" denotes, for example, microorganisms,
yeast cells, insect cells, and mammalian cells, that can be, or
have been, used as recipients of an rLV helper construct, an rLV
vector plasmid, an accessory function vector, or other transfer
DNA. The term includes the progeny of the original cell which has
been transfected. Thus, a "host cell" as used herein generally
refers to a cell which has been transfected with an exogenous DNA
sequence. It is understood that the progeny of a single parental
cell may not necessarily be completely identical in morphology or
in genomic or total DNA complement as the original parent, due to
natural, accidental, or deliberate mutation.
[0052] An accessory function vector generally refers to a nucleic
acid that includes a sequence providing an accessory or helper
function. An accessory function vector can be transfected into a
host cell, and the vector can provide or encode protein(s) that
function to support rLV vector virion production in/by the host
cell. An accessory function vector can be in the form of a plasmid,
phage, transposon, cosmid, episome or integrated in the genome of
the host cell.
[0053] The term "transfection" is used to refer to the uptake of
foreign nucleic acid (e.g., DNA) by a cell, and a cell has been
"transfected" when exogenous nucleic acid (e.g., DNA) has been
introduced inside the cell membrane. A number of transfection
techniques are generally known in the art. See, e.g., Graham et al.
(1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning,
a laboratory manual, Cold Spring Harbor Laboratories, New York,
Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier,
and Chu et al. (1981) Gene 13:197. Such techniques can be used to
introduce one or more exogenous DNA moieties into suitable host
cells.
[0054] As used herein, the term "cell line" refers to a population
of cells capable of continuous or prolonged growth and division in
vitro. Often, cell lines are clonal populations derived from a
single progenitor cell. It is further known in the art that
spontaneous or induced changes can occur in karyotype during
storage or transfer of such clonal populations. Therefore, cells
derived from the cell line referred to may not be precisely
identical to the ancestral cells or cultures, and the cell line
referred to includes such variants.
[0055] Cells and cell lines appropriate for serum free growth in
suspension in accordance with the invention methods include
mammalian cells. Exemplary non-limiting cells include, for example,
HEK 293T (ATCC); HEK293F (Life Technologies); HEK293(ATCC); 293S
(ATCC), BHK (ATCC), BHK-21 (ATCC), CHO (ATCC), CHO/dhFr- (ATCC)1,
and CHO K1 (ATCC) cells.
[0056] Serum free cell growth medium for use in accordance with the
invention methods are available commercially or can be made.
Non-limiting exemplary serum free growth medium include, for
example, FreeStyle.TM.293 (Gibco.RTM., Life Technologies), DMEM/F12
(Gibco.RTM., Life Technologies), SFM4Transfx-293 (HyClone.TM.,
ThermoScientific), CDM4HEK293 (HyClone.TM., ThermoScientific),
StemPro-34SFM (Gibco.RTM., Life Technologies), FreeStyle F17
(Gibco.RTM., Life Technologies), 293SFM II (Gibco.RTM., Life
Technologies), and CD293 (Gibco.RTM., Life Technologies) media.
[0057] In the methods of the invention, a treatment or method step
can be used to reduce or decrease the amount of a nucleic acid
impurity. In particular embodiments, a nuclease is used to reduce
or decrease the amount of a nucleic acid impurity in harvested or a
preparation of recombinant viral vectors. In particular
embodiments, a nuclease is an endonuclease, such as benzonase, an
exonuclease, or a combination thereof. In particular embodiments, a
nuclease is a deoxyribonuclease, a ribonuclease, or a combination
thereof. In particular embodiments, a nuclease is a DNase.
[0058] In the methods of the invention, one or more column
chromatography steps are performed. Various substrates are suitable
as a resin or media (stationary phase) for column chromatography.
Such resin or media (stationary phase) include charge based (ion
exchange) or affinity resin or media.
[0059] In particular embodiments, ion exchange column
chromatography is anion (strong or weak) exchange column
chromatography, or cation (strong or weak) exchange column
chromatography. In more particular embodiments, an ion exchange
column is a quarternized polyehtyleneimine based resin or media; or
a quaternary amine based resin or media. In further more particular
embodiments, an ion exchange column is a polyethyleneimine based
resin; a Diethylaminoethyl (DEAE) based resin; or a
Diethylaminopropyl based resin.
[0060] In particular embodiments, an affinity column is a
Sulphopropyl based resin or media, or a carboxymethyl based resin
or media. In additional particular embodiments, an affinity column
is a multifunctional chromatography resin or media; a Metal Chelate
Affinity resin or media; a heparin based resin or media, or a group
specific affinity resin or media.
[0061] Additional resins or media suitable for column
chromatography in the methods of the invention include an
Hydroxyapatite ((Ca.sub.5(PO.sub.4).sub.3OH).sub.2) based resin or
media; a multimodal weak cation exchange resin or media;
N-benzyl-n-methyletheanolamine based resin or media; or an
Octylamine based resin or media.
[0062] In the methods of the invention, solutions are used, such as
binding, washing and eluting solutions. The terms are used for
convenience to refer to the purpose of the solution within the
context of chromatography.
[0063] Such solutions can optionally include ingredients such as
polyethylene glycol (PEG). Such solutions also can optionally
include ingredients such as salts. In addition, such solutions can
optionally include ingredients such as buffering agents (tris- or
phosphate-buffered). Furthermore, such solutions can include
ingredients such as chelating agents, for example, EDTA.
[0064] In particular embodiments, the amount of PEG in a binding
solution is from about 0% to 10% weight/volume, or from about 0% to
5% weight/volume, or from about 0% to 2% weight/volume. In
particular embodiments, the amount of PEG in a washing solution is
from about 1% to 10% weight/volume, or from about 1% to 5%
weight/volume, or from about 1% to 2% weight/volume. In particular
embodiments, the amount of PEG in an elution solution is from about
0% to 20% weight/volume.
[0065] In particular embodiments, PEG in a binding, washing or
eluting solution has a molecular weight from about 2,000 kDa to
about 40,000 kDa. In other particular embodiments, PEG in a
binding, washing or eluting solution has a molecular weight from
about 2,000 kDa to about 10,000 kDa
[0066] In particular embodiments, a salt comprises or consists of
sodium chloride (NaCl), potassium chloride (KCl), or calcium
chloride. In particular embodiments, the amount of a salt in a
binding, washing or eluting solution is from about 20 mM to about 1
M. In more particular embodiments, the amount of a salt in a
binding solution, is from about 20 mM to about 200 mM (such as
about 100 mM). In more particular embodiments, the amount of a salt
in a washing solution, is from about 20 mM to about 200 mM (such as
100 mM). In more particular embodiments, the amount of a salt in an
eluting solution is from about 200 mM to about 1 M (such as 200 mM,
250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mM) or from about
500-1,000 mM, or 600-800 mM.
[0067] In the methods of the invention, filters may be employed.
Filters can be of various pore size diameters. The pore size
diameter can conveniently be represented by a numerical value.
Exemplary pore sizes range from about 0.20-0.5 um (micron).
Additional exemplary pore sizes range from about 0.20 um (micron)
to about 0.22 um (micron), or more particularly about 0.22 um
(micron). Further exemplary pore sizes range from about 0.22 um
(micron) to about 0.30 um (micron), or about 0.30 um (micron) to
about 0.45 um (micron) pore diameter, or more particularly about
0.45 um (micron).
[0068] The invention methods provide an increase in rLV titers
during large scale production while reducing, decreasing or
eliminating, rLV vector related impurities (e.g. rLV associated
nucleic acid impurities) contained within purified stocks of rLV
virions, with minimal loss to rLV vector particles or virions
contained therein Impurities include protein, nucleic acid (DNA,
RNA), debris, and other material distinct from rLV vector particles
or virions that may be present. Invention methods serve to increase
the amount of rLV vector particles/virions while reducing
decreasing or eliminating impurities.
[0069] In particular embodiments, a method of the invention results
in viral vector produced at approximately 5.times.10.sup.5
infectiousunits (IU)/ml, or more. In particular embodiments, a
method of the invention results in viral vector produced at
approximately 6.times.10.sup.6 infectious units (IU)/ml, or more.
In particular embodiments, a method of the invention results in
viral vector produced at approximately 3.times.10.sup.8 infectious
units (IU)/ml.
[0070] As used herein, the term "about" or "approximately" when
used in reference to a quantity or unit measure refers to a range
of statistical deviation acceptable for the represented numerical
values. Typically, the range is about +/-10%, or +/-5% of the
represented numerical value.
[0071] As disclosed herein, a recombinant (viral) vector may
include a nucleic acid, such as a transgene. A "nucleic acid"
sequence refers to a DNA or RNA sequence. The term captures
sequences that include any of the known base analogues of DNA and
RNA such as, but not limited to 4-acetylcytosine,
8-hydroxy-N6-methyladenosine, aziridinylcytosine,
pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil,
5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethylaminomethyluracil-, dihydrouracil, inosine,
N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
oxybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
Buracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.
[0072] A "coding sequence" or a sequence which "encodes" a selected
polypeptide, is a nucleic acid molecule which is transcribed (in
the case of DNA) and translated (in the case of mRNA) into a
polypeptide in vivo when placed under the control of appropriate
regulatory sequences. The boundaries of the coding sequence are
determined by a start codon at the 5' (amino) terminus and a
translation stop codon at the 3' (carboxy) terminus A transcription
termination sequence may be located 3' to the coding sequence.
[0073] The term DNA "control sequences" refers collectively to
promoter sequences, polyadenylation signals, transcription
termination sequences, upstream regulatory domains, origins of
replication, internal ribosome entry sites ("IRES"), enhancers, and
the like, which collectively provide for the replication,
transcription and translation of a coding sequence in a recipient
cell. Not all of these control sequences need always be present so
long as the selected coding sequence is capable of being
replicated, transcribed and translated in an appropriate host
cell.
[0074] The term "promoter" is used herein in its ordinary sense to
refer to a nucleotide region comprising a DNA regulatory sequence,
wherein the regulatory sequence is derived from a gene which is
capable of binding RNA polymerase and initiating transcription of a
downstream (3'-direction) coding sequence. Transcription promoters
can include "inducible promoters" (where expression of a
polynucleotide sequence operably linked to the promoter is induced
by an analyte, cofactor, regulatory protein, etc.), "repressible
promoters" (where expression of a polynucleotide sequence operably
linked to the promoter is induced by an analyte, cofactor,
regulatory protein, etc.), and "constitutive promoters."
[0075] "Operably linked" refers to an arrangement of elements
wherein the components so described are configured so as to perform
their usual function. Thus, control sequences operably linked to a
coding sequence are capable of effecting the expression of the
coding sequence. The control sequences need not be contiguous with
the coding sequence, so long as they function to direct the
expression thereof. Thus, for example, intervening untranslated yet
transcribed sequences can be present between a promoter sequence
and the coding sequence and the promoter sequence can still be
considered "operably linked" to the coding sequence.
[0076] For the purpose of describing the relative position of
nucleotide sequences in a particular nucleic acid molecule
throughout the instant application, such as when a particular
nucleotide sequence is described as being situated "upstream,"
"downstream," "3'," or "5'" relative to another sequence, it is to
be understood that it is the position of the sequences in the
"sense" or "coding" strand of a DNA molecule that is being referred
to as is conventional in the art.
[0077] The term "heterologous" as it relates to nucleic acid
sequences such as coding sequences and control sequences, denotes
sequences that are not normally joined together, and/or are not
normally associated with a particular cell. Thus, a "heterologous"
region of a nucleic acid construct or a vector is a segment of
nucleic acid within or attached to another nucleic acid molecule
that is not found in association with the other molecule in nature.
For example, a heterologous region of a nucleic acid construct
could include a coding sequence flanked by sequences not found in
association with the coding sequence in nature. Another example of
a heterologous coding sequence is a construct where the coding
sequence itself is not found in nature (e.g., synthetic sequences
having codons different from the native gene). Similarly, a cell
transformed with a construct which is not normally present in the
cell would be considered heterologous for purposes of this
invention. Allelic variation or naturally occurring mutational
events do not give rise to heterologous DNA, as used herein.
[0078] A "therapeutic molecule" in one embodiment is a peptide or
protein that may alleviate or reduce symptoms that result from an
absence or defect in a protein in a cell or subject. Alternatively,
a "therapeutic" peptide or protein encoded by a transgene is one
that confers a benefit to a subject, e.g., to correct a genetic
defect, to correct a gene (expression or functional) deficiency, or
an anti-cancer effect. Accordingly, a transgene comprising the
heterologous nucleic acid can encode a number of useful products.
These can include siRNA, antisense molecules, and miRNAs for
example.
[0079] Transgenes can encode hormones and growth and
differentiation factors including, without limitation, insulin,
glucagon, growth hormone (GH), parathyroid hormone (PTH), growth
hormone releasing factor (GRF), follicle stimulating hormone (FSH),
luteinizing hormone (LH), human chorionic gonadotropin (hCG),
vascular endothelial growth factor (VEGF), angiopoietins,
angiostatin, granulocyte colony stimulating factor (GCSF),
erythropoietin (EPO), connective tissue growth factor (CTGF), basic
fibroblast growth factor (bFGF), acidic fibroblast growth factor
(aFGF), epidermal growth factor (EGF), transforming growth factor
.alpha. (TGF.alpha.), platelet-derived growth factor (PDGF),
insulin growth factors I and II (IGF-I and IGF-II), any one of the
transforming growth factor .beta. superfamily, including TGF.beta.,
activins, inhibins, or any of the bone morphogenic proteins (BMP)
BMPs 1-15, any one of the heregluin/neuregulin/ARIA/neu
differentiation factor (NDF) family of growth factors, nerve growth
factor (NGF), brain-derived neurotrophic factor (BDNF),
neurotrophins NT-3 and NT4/5, ciliary neurotrophic factor (CNTF),
glial cell line derived neurotrophic factor (GDNF), neurturin,
agrin, any one of the family of semaphorins/collapsins, netrin-1
and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin,
sonic hedgehog and tyrosine hydroxylase.
[0080] Other useful transgene products include proteins that
regulate the immune system including, without limitation, cytokines
and lymphokines such as thrombopoietin (TPO), interleukins (IL)
IL-1 through IL-17, monocyte chemoattractant protein, leukemia
inhibitory factor, granulocyte-macrophage colony stimulating
factor, Fas ligand, tumor necrosis factors .alpha. and .beta.,
interferons .alpha., .beta., and .gamma., stem cell factor,
flk-2/flt3 ligand. Gene products produced by the immune system are
also useful in the invention. These include, without limitations,
immunoglobulins IgG, IgM, IgA, IgD and IgE, chimeric
immunoglobulins, humanized antibodies, single chain antibodies, T
cell receptors, chimeric T cell receptors single chain T cell
receptors (e.g. Kalos et al 2011; Porter et al 2011), G
protein-coupled receptors (GPCRs) such as CCR5, class I and class
II MHC molecules, as well as engineered immunoglobulins and MHC
molecules. Useful gene products also include regulatory proteins
such as complement regulatory proteins, membrane cofactor protein
(MCP), decay accelerating factor (DAF), CR1, CF2 and CD59.
[0081] Other useful gene products include those that can correct in
born errors of metabolism. Such transgenes can encode for example,
carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate
synthetase, arginosuccinate lyase, arginase, fumarylacetacetate
hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin,
glucose-6-phosphatase, porphobilinogen deaminase, blood clotting
factors such as Factor V, Factor VIIa, Factor VIII, Factor IX,
Factor X, Factor XIII, or protein C, cystathione beta-synthase,
branched chain ketoacid decarboxylase, albumin, isovaleryl-coA
dehydrogenase, propionyl CoA carboxylase, methyl malonyl CoA
mutase, glutaryl CoA dehydrogenase, insulin, beta-glucosidase,
pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase,
glycine decarboxylase, H-protein, T-protein, a cystic fibrosis
transmembrane regulator (CFTR) sequence, and a dystrophin cDNA
sequence.
[0082] Further useful gene products include those that can provide
for a defective, deficient or missing function or activity, for
example, an antibody, retinal pigment epithelium-specific 65 kDa
protein (RPE65), erythropoietin, LDL receptor, lipoprotein lipase,
ornithine transcarbamylase, .beta.-globin, .alpha.-globin,
spectrin, .alpha.-antitrypsin, adenosine deaminase (ADA), a metal
transporter (ATP7A or ATP7), sulfamidase, an enzyme involved in
lysosomal storage disease (ARSA), hypoxanthine guanine
phosphoribosyl transferase, .beta.-25 glucocerebrosidase,
sphingomyelinase, lysosomal hexosaminidase, branched-chain keto
acid dehydrogenase, a hormone, a growth factor (e.g., insulin-like
growth factors 1 and 2, platelet derived growth factor, epidermal
growth factor, nerve growth factor, neurotrophic factor-3 and -4,
brain-derived neurotrophic factor, glial derived growth factor,
transforming growth factor .alpha. and .beta., etc.), a cytokine
(e.g., .alpha.-interferon, .beta.-interferon, interferon-.gamma.,
interleukin-2, interleukin-4, interleukin 12,
granulocyte-macrophage colony stimulating factor, lymphotoxin,
etc.), a suicide gene product (e.g., herpes simplex virus thymidine
kinase, cytosine deaminase, diphtheria toxin, cytochrome P450,
deoxycytidine kinase, tumor necrosis factor, etc.), a drug
resistance protein (e.g, that provides resistance to a drug used in
cancer therapy), a tumor suppressor protein (e.g., p53, Rb, Wt-1,
NF1, Von Hippel-Lindau (VHL), adenomatous polyposis coli (APC)), a
peptide with immunomodulatory properties, a tolerogenic or
immunogenic peptide or protein Tregitopes, or hCDR1, insulin,
glucokinase, guanylate cyclase 2D (LCA-GUCY2D), Rab escort protein
1 (Choroideremia), LCA 5 (LCA-Lebercilin), ornithine ketoacid
aminotransferase (Gyrate Atrophy), Retinoschisin 1 (X-linked
Retinoschisis), USH1C (Usher's Syndrome 1C), X-linked retinitis
pigmentosa GTPase (XLRP), MERTK (AR forms of RP: retinitis
pigmentosa), DFNB1 (Connexin 26 deafness), ACHM 2, 3 and 4
(Achromatopsia), PKD-1 or PKD-2 (Polycystic kidney disease), TPP1,
CLN2, gene deficiencies causative of lysosomal storage diseases
(e.g., sulfatases, N-acetylglucosamine-1-phosphate transferase,
cathepsin A, GM2-AP, NPC1, VPC2, Sphingolipid activator proteins,
etc.), one or more zinc finger nucleases for genome editing, or
donor sequences used as repair templates for genome editing.
[0083] Transgenes also can encode a tumor associated antigens
(TAAs). Non-limiting TAAs include: tumor-associated testis-specific
antigen (e.g., MAGE, BAGE, and GAGE), melanocyte differentiation
antigen (e.g., tyrosinase, Melan-A/MART-1), CDK4, MUM-1,
beta-catenin, gp100/pmel 17, TRP-1, TRP-2, an MITF, MITF-A and
MITF-M (King, et al. (1999). Am J Pathol 155:731). Additional
non-limiting examples of TAAs expressed by tumors include melanoma
GP75, Annexin I, Annexin II, adenosine deaminase-binding protein
(ADAbp), PGP 9.5 (Rode, et al. (1985). Histopathology 9:147),
colorectal associated antigen (CRC)--C017-1A/GA733, Ab2 BR3E4,
CI17-1A/GA733, Hsp70 (Chen, et al. (2002). Immunol Lett 84:81),
Hsp90, Hsp96, Hsp105, Hsp110, HSPPC-96 (Caudill, M. M. and Z. Li
(2001). Expert Opin Biol Ther 1:539), stress protein gp96 (Heike et
al. (2000). Int J Can 86:489), gp96-associated cellular peptides,
G250, Dipeptidyl peptidase IV (DPPIV), Mammaglobin (Tanaka, et al.
(2003). Surgery 133:74), thyroglobulin, STn (Morse, M. A. (2000).
Curr Opin Mol Ther 2:453), Carcinoembryonic Antigen (CEA),
Carcinoembryonic Antigen (CEA) epitope CAP-1, Carcinoembryonic
Antigen (CEA) epitope CAP-2, etv6, am11, Prostate Specific Antigen
(PSA), PSA epitopes PSA-1, PSA-2, PSA-3 (Correale, et al. (1998). J
Immunol 161:3186), Ad5-PSA, Parathyroid-hormone-related protein
(PTH-rP), EGFR (Plunkett, et al. (2001). J Mammary Gland Biol
Neoplasia 6:467), PLU1 (Plunkett, et al. (2001). J Mammary Gland
Biol Neoplasia 6:467), Oncofetal antigen-immature laminin receptor
(OFA-iLR), MN/CA IX (CA9) (Shimizu et al., (2003). Oncol. Rep.
September-October; 10:1307), HP59, Cytochrome oxidase 1, sp100, msa
(Devine, et al. (1991). Cancer Res 51:5826), Ran GTPase activating
protein, a Rab-GAP (Rab GTPase-activating) protein, PARIS-1 (Zhou,
et al. (2002). Biochem Biophys Res Commun 290:830), T-cell
receptor/CD3-zeta chain, cTAGE-1, SCP-1, Glycolipid antigen-GM2,
GD2 or GD3, GM3 (Bada, et al. (2002). Hum Exp Toxicol 21:263),
FucosylGM1, Glycoprotein (mucin) antigens-Tn, Sialyl-Tn (Lundin, et
al. (1999). Oncology 57:70), TF and Mucin-1 (Mukherjee, et al.
(2003). J Immunother 26:47), CA125 (MUC-16) (Reinartz, et al.
(2003). Cancer Res 63:3234), a MAGE family antigen, GAGE-1,2, BAGE,
RAGE, LAGE-1 (Eichmuller, et al. (2003). Int J Cancer 104:482)
(Chen, et al. (1998). Proc Natl Acad Sci USA 95:6919), GnT-V
(Murata, et al. (2001). Dis Colon Rectum 44:A2-A4), MUM-1
(Kawakami, et al. (1996). Keio J Med 45:100), EP-CAM/KSA (Ullenhag,
et al. (2003). Clin Cancer Res 9:2447), CDK4, a MUC family antigen,
HER2/neu, ErbB-2/neu, p21ras, RCAS1, .alpha.-fetoprotein,
E-cadherin, .alpha.-catenin, .beta.-catenin and .gamma.-catenin,
NeuGcGM3 (Carr, et al. (2003). J Clin Oncol 21:1015), Fos related
antigen (Luo, et al. (2003). Proc Natl Acad Sci USA 100:8850),
Cyclophilin B (Tamura, et al. (2001). Jpn J Cancer Res 92:762),
RCAS1, S2 (Koga, et al. (2003). Tissue Antigens 61:136), L10a
(Koga, et al. (2003). supra), L10a, Telomerase rt peptide (Wang, et
al. (2001). Oncogene 20:7699), cdc27, fodrin, p120ctn, PRAME,
GA733/EoCam (Ross, et al. (1986). Biochem Biophys Res Commun
135:297), NY-BR-1, NY-BR-2 NY-BR-3, NY-BR-4 NY-BR-5, NY-BR-6
NY-BR-7 (Jager, et al. (2001). Cancer Res 61:2055), NY-ESO-1,
L19H1, MAZ (Daheron, et al. (1998). Leukemia 12:326), PINCH
(Greiner, et al. (2000). Exp Hematol 28:1413), PRAME (Ikeda, et al.
(1997) Immunity 6:199), Prp1p/Zer1p, WT1 (Oka, et al. (2002). Curr
Cancer Drug Targets 2:45), adenomatous polyposis coli protein
(APC), PHF3, LAGE-1, SART3 (Miyagi, et al. (2001). Clin Cancer Res
7:3950), SCP-1 (Jager, et al. (2002). Cancer Immun 2:5), SSX-1,
SSX-2, SSX-4, TAG-72 (Buchsbaum, et al. (1999). Clin Cancer Res
5(10 Suppl): 3048s-3055s), TRAG-3 (Chen, et al. (2002). Lung Cancer
38:101), MBTAA (Basu, et al. (2003). Int J Cancer 105:377), a Smad
tumor antigen, lmp-1, HPV-16 E7, c-erbB-2, EBV-encoded nuclear
antigen (EBNA)-1, Herpes simplex thymidine kinase (HSVtk),
alternatively spliced isoform of XAGE-1 (L552S; Wang, (2001).
Oncogene 20:7699), TGF beta RII frame shift mutation (Saeterdal, et
al. (2001). Proc Natl Acad Sci USA 98:13255), BAX frame shift
mutation (Saeterdal, et al. (2001). Proc Natl Acad Sci USA
98:13255).
[0084] Transgenes additionally can encode a gene product, such as
CAIX, CD19, CD20, CD20, CD22, CD30, CD33, CD44v7/8, CEA, EGF-RIII
(epidermal growth factor receptor variant 3) EGP-2, erb-B2, erb-B2,
3, 4, FBP, fetal acetycholine receptor, GD2, Her2/neu, IL-13R-a2,
KDR, k-light chain, LeY, L1 cell adhesion molecule, MAGE-A1,
mesothelin, MUC1, NKG2D, oncofetal antigen (h5T4), PSCA,
prostate-specific membrane antigen (PSMA), Prostatic Acid
Phosphatase (PAP), Prostate epithelium-derived Ets transcription
factor (PDEF), mAb IgE targeted TAA, TAG-72 and VEGF-R2.
[0085] Alternatively, transgenes can include siRNA, antisense
molecules, and miRNAs for example. Clinically useful lentiviral
vectors may also express an antisense gene directed against the
human immunodeficiency virus (HIV) (Levine et al 2006) and other
important human pathogens. These and other useful applications of
rLV and related vectors have been recently reviewed and cited by
Naldini (2011).
[0086] Antisense genes that can be included in an rLV vector can
inhibit expression of: huntingtin (HTT) gene, a gene associated
with dentatorubropallidolusyan atropy (e.g., atrophin 1, ATN1);
androgen receptor on the X chromosome in spinobulbar muscular
atrophy, human Ataxin-1, -2, -3, and -7, Ca.sub.v2.1 P/Q
voltage-dependent calcium channel is encoded by the (CACNA1A),
TATA-binding protein, Ataxin 8 opposite strand, also known as
ATXN8OS, Serine/threonine-protein phosphatase 2A 55 kDa regulatory
subunit B beta isoform in spinocerebellar ataxia (type 1, 2, 3, 6,
7, 8, 12 17), FMR1 (fragile X mental retardation 1) in fragile X
syndrome, FMR1 (fragile X mental retardation 1) in fragile
X-associated tremor/ataxia syndrome, FMR1 (fragile X mental
retardation 2) or AF4/FMR2 family member 2 in fragile XE mental
retardation; Myotonin-protein kinase (MT-PK) in myotonic dystrophy;
Frataxin in Friedreich's ataxia; a mutant of superoxide dismutase 1
(SOD1) gene in amyotrophic lateral sclerosis; a gene involved in
pathogenesis of Parkinson's disease and/or Alzheimer's disease;
apolipoprotein B (APOB) and proprotein convertase subtilisin/kexin
type 9 (PCSK9), hypercoloesterolemia; HIV Tat, human
immunodeficiency virus transactivator of transcription gene, in HIV
infection; HIV TAR, HIV TAR, human immunodeficiency virus
transactivator response element gene, in HIV infection; C--C
chemokine receptor (CCR5) in HIV infection; Rous sarcoma virus
(RSV) nucleocapsid protein in RSV infection, liver-specific
microRNA (miR-122) in hepatitis C virus infection; p53, acute
kidney injury or delayed graft function kidney transplant or kidney
injury acute renal failure; protein kinase N3 (PKN3) in advance
recurrent or metastatic solid malignancies; LMP2, LMP2 also known
as proteasome subunit beta-type 9 (PSMB 9), metastatic melanoma;
LMP7, also known as proteasome subunit beta-type 8 (PSMB 8),
metastatic melanoma; MECL1 also known as proteasome subunit
beta-type 10 (PSMB 10), metastatic melanoma; vascular endothelial
growth factor (VEGF) in solid tumors; kinesin spindle protein in
solid tumors, apoptosis suppressor B-cell CLL/lymphoma (BCL-2) in
chronic myeloid leukemia; ribonucleotide reductase M2 (RRM2) in
solid tumors; Furin in solid tumors; polo-like kinase 1 (PLK1) in
liver tumors, diacylglycerol acyltransferase 1 (DGAT1) in hepatitis
C infection, beta-catenin in familial adenomatous polyposis; beta2
adrenergic receptor, glaucoma; RTP801/Redd1 also known as DAN
damage-inducible transcript 4 protein, in diabetic macular oedma
(DME) or age-related macular degeneration; vascular endothelial
growth factor receptor I (VEGFR1) in age-related macular
degeneration or choroidal neivascularization, caspase 2 in
non-arteritic ischaemic optic neuropathy; Keratin 6A N17K mutant
protein in pachyonychia congenital; influenza A virus genome/gene
sequences in influenza infection; severe acute respiratory syndrome
(SARS) coronavirus genome/gene sequences in SARS infection;
respiratory syncytial virus genome/gene sequences in respiratory
syncytial virus infection; Ebola filovirus genome/gene sequence in
Ebola infection; hepatitis B and C virus genome/gene sequences in
hepatitis B and C infection; herpes simplex virus (HSV) genome/gene
sequences in HSV infection, coxsackievirus B3 genome/gene sequences
in coxsackievirus B3 infection; silencing of a pathogenic allele of
a gene (allele-specific silencing) like torsin A (TOR1A) in primary
dystonia, pan-class I and HLA-allele specific in transplant; mutant
rhodopsin gene (RHO) in autosomal dominantly inherited retinitis
pigmentosa (adRP); or the inhibitory nucleic acid binds to a
transcript of any of the foregoing genes or sequences.
[0087] By "isolated" when referring to a nucleotide sequence, is
meant that the indicated molecule is present in the substantial
absence of other biological macromolecules of the same type. Thus,
an "isolated nucleic acid molecule which encodes a particular
polypeptide" refers to a nucleic acid molecule which is
substantially free of other nucleic acid molecules that do not
encode the subject polypeptide; however, the molecule may include
some additional bases or moieties which do not deleteriously affect
the basic characteristics of the composition.
[0088] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described herein.
[0089] All applications, publications, patents and other
references, GenBank citations and ATCC citations disclosed herein
are incorporated by reference in their entirety. In case of
conflict, the specification, including definitions, will
control.
[0090] All of the features disclosed herein may be combined in any
combination. Each feature disclosed in the specification may be
replaced by an alternative feature serving a same, equivalent, or
similar purpose. Thus, unless expressly stated otherwise, disclosed
features (e.g., a recombinant vector (e.g., rLV) vector, or
recombinant virus particle are an example of a genus of equivalent
or similar features.
[0091] As used herein, the singular forms "a", "and," and "the"
include plural referents unless the context clearly indicates
otherwise. Thus, for example, reference to "a polynucleotide"
includes a plurality of such polynucleotides, reference to "a
vector" includes a plurality of such vectors, and reference to "a
virus" or "particle" includes a plurality of such
virions/particles.
[0092] As used herein, all numerical values or numerical ranges
include integers within such ranges and fractions of the values or
the integers within ranges unless the context clearly indicates
otherwise. Thus, to illustrate, reference to at least 1-10%%
identity, includes 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, as well
as 1.1%, 1.2%, 1.3% 1.4%, 1.5%, etc., 2.1%, 2.2%, 2.3%, 2.4%, 2.5%,
etc., and so forth.
[0093] Reference to a number with more (greater) or less than
includes any number greater or less than the reference number,
respectively. Thus, for example, a reference to less than 40,000,
includes 39,999, 39,998, 39,997, etc. all the way down to the
number one (1); and less than 100, includes 99, 98, 97, etc. all
the way down to the number one (1).
[0094] As used herein, all numerical values or ranges include
fractions of the values and integers within such ranges and
fractions of the integers within such ranges unless the context
clearly indicates otherwise. Thus, to illustrate, reference to a
numerical range, such as 2,000-40,000 includes 2,000; 3,000; 4,
000; 5,000, 6,000, etc. as well as 2,100; 3,100; 4,100; 5,100;
6,100; etc., and so forth. Reference to a range of 20-100 therefore
includes 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, etc., up to and including 100, as well as
21.1, 21.2, 21.3, 21.4, 21.5, etc., 22.1, 22.2, 22.3, 22.4, 22.5,
etc., and so forth.
[0095] Reference to a series of ranges includes ranges which
combine the values of the boundaries of different ranges within the
series. Thus, to illustrate reference to a series of ranges of
20-100 or 100-1,000 (e.g., 20 mM-100 mM; or 100 mM-1M) includes
20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200,
200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, etc.
[0096] The invention is generally disclosed herein using
affirmative language to describe the numerous embodiments and
aspects. The invention also specifically includes embodiments in
which particular subject matter is excluded, in full or in part,
such as substances or materials, method steps and conditions,
protocols, or procedures. For example, in certain embodiments or
aspects of the invention, materials and/or method steps are
excluded. Thus, even though the invention is generally not
expressed herein in terms of what the invention does not include
aspects that are not expressly excluded in the invention are
nevertheless disclosed herein.
[0097] A number of embodiments of the invention have been
described. Nevertheless, one skilled in the art, without departing
from the spirit and scope of the invention, can make various
changes and modifications of the invention to adapt it to various
usages and conditions. Accordingly, the following examples are
intended to illustrate but not limit the scope of the invention
claimed.
EXAMPLES
Example 1
[0098] Lentivirus vectors are typically produced by transfection
methods using calcium phosphate precipitation method in adherent
cell culture system (3, 4). However, when manufacturing
lentivectors for clinical application, particularly for late stages
of clinical applications, it is critical to manufacture the vectors
in a serum free suspension production system which can be scaled up
to ensure the quantity of the vector production and enhance the
safety profile of the vector manufactured.
[0099] To overcome the existing limitations of currently available
Lentivector production systems, disclosed herein is a scalable
serum free, suspension cell culture system producing high titer
lentivectors. The first step in this new scalable production system
is the development of a cell line that can be cultured in serum
free condition and produces high levels of lentivectors. We
hypothesized that adapting HEK293T cells to serum free suspension
culture should be feasible as these cells are currently widely used
in producing viral vectors. To achieve this goal, we designed a
step wise adaption protocol and systemically evaluated several
commercially available serum-free cell culture media and the
ability of HEK 293T cells to grow robustly in these media. One
media (CD293) was found to outperform all of the others tested in
supporting non-aggregated, healthy cell growth after adaption with
fast growth rate (FIG. 2, Panel A). The optimized cell culture
condition support high cell density and consistent cell growth
(FIG. 2, Panel B and C). The adapted cells have been cultured under
serum free suspension culture conditions for several months, and a
research cell bank has been developed.
[0100] Lentiviral vectors have been produced by calcium phosphate
precipitation co-transfection of 2 to 5 plasmids into target cells.
While the latest generation of the multiplasmid production system
(self in activation system) has been shown to be versatile in
producing lentivectors with a reduced risk of generating
replication competent lentivirus, the calcium co-precipitation
imposes a limitation on large-scale manufacture. Calcium phosphate
co-precipitation of DNA, developed over 40 years ago (5), works
well for adherent cell culture in research scale. However, it does
not provide efficient levels of transfection in serum free
suspension cell cultures and is very difficult to scale up.
[0101] Polyethylenimine (PEI) is commercially available and was
tested for efficient introduction of plasmid DNA into the newly
adapted HEK 293T cells (6). Initial experiments revealed that the
media selected for serum free cell culture did not support any
transfection using PEI as transfection reagent, efforts were then
directed to identify a serum free media that will support
transfection of the cells using PEI. While one particular media
type screened supported transfection (FIG. 3 Panel D), this media
did not support suspension growth of the cells in long term cell
culture (data not shown). We therefore design and developed a two
step system to produce lentivectors in serum free suspension:
First, cells are grown in the media that supports robust and rapid
growth in suspension under serum free conditions. Second, the cell
culture media is either replaced or mixed with a second media type
that support PEI based transfection. In efforts to optimize PEI
transfection efficiency, several different forms of PEI molecules
from different commercial venders were evaluated. While small
molecular weight linear PEI, such 25-kDa linear PEI has been used
in several cases in manufacture of biological products (6), we
hypothesis that branched PEI may exhibit greater delivery
efficiencies for multiple plasmid transfections, due to the
availability of multiple functional groups per molecule. Among the
different PEI molecules evaluated, a small branched PEI, PEI MAX
(40,000 kDa, Polyscience .com) resulted in the highest transfection
efficiency of the adapated HEK 293T cells in the media identified
(FIG. 4). Optimizing transfection condition using PEI MAX, we
achieved almost one hundred percent cell transfection (FIG. 5 Panel
A). Our preliminary semi-quantitative data indicated that
lentiviral vectors were produced at the level about
1.times.10.sup.6 transduction units per ml in the current
production method. To the best of our knowledge, we believe the
techniques innovated in our lab represent the first truly scalable
lentivector production method in a serum free suspension cell
culture system.
[0102] Further effort was made to improve vector specific
productivity. Parameters, such as cell density at transfection,
total DNA amount used for transfection, methods to prepare DNA/PEI
complex, vector harvest time, were evaluated. The adapted HEK 293 T
cells were cultured in CD293 media (complemented with 4 mM
Glutamine) to the density of 3E+06/ml; the cell culture media was
exchange to SFM4Transfx-293 (complemented with 4 mM Glutamine)
(HyClone.TM., ThermoScientific) by either centrifugation and
re-suspension or using tangential flow filtration technique, cell
density was adjusted to 1.5E+06 cells per ml and incubated
incubator. For the spinner flask cell culture, 130 RPM/min and 8%
CO.sub.2 were used. Total of 12 ug DNA was used to transfect
1.5E+06 cells and the DNA molar ratio of the four plasmids was
1:1:1:1. Polyethyleneimine"MAX" (Polysciences.com) was prepared
using Tris buffered solution at concentration of 1 mg/ml and pH
adjusted to 7.15. Mixture of DNA was added to PEI solution with
mass ratio of 1:1, and the solution was mixed gently and further
incubated shortly; the mixed DNA/PEI cocktail was further diluted
using 5 mM Tris solution, pH 7.15; the final volume of the diluted
DNA/PEI solution is 1/15 of the media of the cell culture to be
transfected. The DNA/PEI solution was then added to the suspension
cell culture at the volume ration of 1/15, the cell culture media
was then harvested at 48 hours, 72 hours and 96 hours post
transfection. HEK 293 cells were then transduced with the harvested
cell culture harvests and analyzed for eGFP expression using FACS.
Higher vector production was observed, more than 6E+06 TU/ml were
produced in from plates and spinner flasks, a more scalable cell
culture platform.
[0103] Column chromatography techniques are widely used in industry
to purify large scale biological materials. Lentivectors are
currently purified either by centrifugation techniques to
precipitate the vectors (3) or by column chromatography techniques
to isolate the particles (6). Column chromatography based processes
address the scale issues in manufacture when using e classic
centrifugation techniques, however, it still remains a challenge to
isolate high purity lentivectors. In the typical anion exchanger
column chromatography, the lentivector harvested was loaded onto
the column, washed with low concentration of slat (such as 100 mM
NaCl), then the vectors were eluted with high salt buffer (650 mM
NaCl to 1M NaCl) (6). A lot of cellular proteins were co-eluted
using this type of chromatography procedures (FIG. 8, Lane 2). We
reported a PEG-modulated column chromatography procedure for
purification of adeno-associated viral vectors (7), the purity of
rAAV vector isolated from the PEG-modulated chromatography was
significantly improved. We hypothesized that PEG-modulated
chromatography should also be applicable to the separation of
Lentivectors from cellular proteins to enhance the vector purity
since the separation is based on the size of the molecules even
though the resins used are not size exclusion resins.
[0104] A PEG-modulated column chromatography procedure was
developed using DEAE Sepharose Fast Flow resins. Lentiviral vectors
were recovered from this procedure at more than 95% of transduction
units based on FACS analysis of eGFP expressing cells (FIG. 5),
while the vector purities are improved 20 fold than the purities
using traditional column chromatography (FIGS. 6 and 7). While we
employed a weak anion exchanger resin to develop the PEG-modulated
purification protocol for lentivectors, we believe the principles
behind this protocol should apply to any column chromatography
resins, including affinity resin, strong and weak anion exchangers,
strong and weak cation exchangers and other resins, as long as the
vector binds to the resin. Based on innovative PEG modulated column
chromatography, a complete scalable purification process was
designed and developed. A flow chart of the procedures described
herein is provided in FIG. 1.
[0105] The techniques described above are fully scalable process
steps that will enable manufacture of sufficient quantities of high
quantity rLenti vectors that are needed to support the exciting
clinical applications that are emerging. Surprisingly, the methods
and rLV compositions produced surpasses currently available
manufacturing capacity for lentiviral vectors and satisfies
investigational product quality requirements.
* * * * *