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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
  • C12N5/0662—Stem cells
  • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
  • A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
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  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/1858—Platelet-derived growth factor [PDGF]
  • A61K38/1866—Vascular endothelial growth factor [VEGF]
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/10—Growth factors
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/10—Growth factors
  • C12N2501/15—Transforming growth factor beta (TGF-β)
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/10—Growth factors
  • C12N2501/165—Vascular endothelial growth factor [VEGF]
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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
  • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
  • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
  • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
  • C12N2740/15045—Special targeting system for viral vectors
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  • C12N2810/00—Vectors comprising a targeting moiety
  • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
  • C12N2810/55—Vectors comprising as targeting moiety peptide derived from defined protein from bacteria

Definitions

• CLI Critical limb ischemia
• PAD peripheral artery disease
• the rate of lower limb amputation in the United States has doubled since 1985 with a 4-to 5-fold increase in those over the age of 80. Fewer than half of all CLI patients achieve full mobility after an amputation and only one in four above-the-knee amputees will ever wear a prosthesis.
• the diagnosis comes at a substantial price to the society as the estimated cost of treating CLI is 10 to 20 billion dollars per year in the US alone.
• This disclosure relates to vectors, isolated cells, compositions, and methods for the treatment of peripheral artery disease (PAD), such as critical limb ischemia and associated disorders.
• PID peripheral artery disease
• One aspect of the disclosure relates to a vector comprising or alternatively consisting essentially of, or yet further consisting of a nucleic acid encoding a 165 A isoform VEGF protein and a promoter that regulates expression of the nucleic acid encoding the VEGF.
• Another aspect relates to a vector comprising or alternatively consisting essentially of, or yet further consisting of the polynucleotide of SEQ ID NO: 2.
• the vector is encoded in the sequence set forth in SEQ ID NO: 2.
• a viral packaging system comprising or alternatively consisting essentially of, or yet further consisting of a vector described herein; a packaging plasmid; and an envelope plasmid.
• pseudotyped viral particles produced by transducing a packaging cell line with a viral packaging system as described herein under conditions suitable to package the viral vector.
• the pseudotyped viral particles are isolated from the supernatant.
• isolated cells comprising or alternatively consisting essentially of, or yet further consisting of a nucleic acid encoding a 165 A isoform VEGF protein and a promoter that regulates expression of the nucleic acid encoding the VEGF.
• the cells are marrow stromal cells.
• Method aspects of the disclosure relate to methods for treating critical limb ischemia in a patient in need thereof comprising or alternatively consisting essentially of, or yet further consisting of administering an isolated cell to the patient as described herein.
• a second method aspect relates to a method for promoting wound healing, promoting or increasing the rate of angiogenesis or wound healing, decreasing the size of a wound, or decreasing the time to wound healing in a patient in need thereof comprising or alternatively consisting essentially of, or yet further consisting of administering an isolated cell described herein to the the patient.
• a further method aspect relates to a method for salvaging a limb in a patient with critical limb ischemia comprising or alternatively consisting essentially of, or yet further consisting of administering an isolated cell described herein to the the patient.
• a yet further method aspect relates to a method for increasing vascularization in a patient in need thereof comprising or alternatively consisting essentially of, or yet further consisting of administering an isolated cell described herein to the the patient.
• isolated mesenchymal stem cell expressing the phenotype CD34-/CD45-/CD105+/CD90+/CD73+ and comprising a 165 A VEGF polynucleotide or protein. Further aspects relate to expanded populations of the described isolated mesenchymal stem cells and compositions comprising the isolated mesenchymal stem cells described herein.
• FIG. 1 shows that the overexpression of growth factors (GF) leads to activation of specific signaling pathways in MSCs.
• MSCs were transduced with control lentiviral vectors or those designed to overexpress GF. Over-expression of GF was then confirmed at both mR A and protein levels.
• C Protein levels of GF measured in supernatants of MSCs using ELISA.
• D Activation of ERK1/2 and AKT1/2 was measured by western blot in MSCs as described.
• FIG. 3 demonstrates that over-expression of bFGF and PDGF-B increase the osteogenic differentiation of MSCs, while TGF- ⁇ inhibits.
• FIG. 4 shows that over-expression of PDGF-B or TGF- ⁇ inhibits the adipogenic differentiation of MSCs.
• Transduced MSCs were cultured in adipogenic medium for 21 days.
• D and
• FIG. 5 shows that TGF- ⁇ induces morphologic alterations in MSCs.
• A Changes in morphology of MSCs over-expressing growth factors were analyzed using phalloidin to stain actin protein (red). Cells over-expressing bFGF or VEGF did not differ from control cells. In contrast, MSCs over-expressing PDGF-B overlap and create extended protrusions. MSCs over-expressing TGF-bl were more flattened and larger than control cells and presented abundant actin bundles characteristic of stress fibers. These changes were evident 3 days after transduction.
• B and
• FIG. 6 shows that over-expression of bFGF and VEGF enhance migration of endothelial cells.
• FIG. 7 shows the VEGF mRNA (FIG. 7A) and protein (FIG. 7B) levels in MSC cells tranduced with the viral vector as described in Example 2.
• FIG. 8 demonstrates that engineering MSCs to produce VEGF does not affect their proliferation rate or differentiative capacity, in established MSC potency assays.
• A) Proliferation of MSC over-expressing GF was measured by counting cells using trypan blue exclusion dye in a hemocytometer, as described (n 3).
• FIG. 12 shows that MSC/VEGF promotes vascular repair in vivo.
• Laser Doppler perfusion imaging was used to assess the ratio blood flow in the healthy versus affected legs. Mean group values ⁇ SD are shown. Asterisks denote significant difference (p ⁇ 0.05).
• FIG. 13 shows that no edema is caused by MSC/VEGF.
• FIG. 14 shows an ELISA which was used to measure VEGF expression from MSC/VEGF after induction with different levels of doxycycline. VEGF production was above 5000 pg/ml per 100,000 cells at all dox levels tested (50 ng per one million cells in 24 hours).
• FIG. 15 shows IND-enabling studies to be completed at the level of GLP using the MSC/VEGF test lot.
• MSCs engineered to express luciferase were evaluated for the duration of retention, and injected at increasing doses.
• the scale shows correlation of signal to cell number to evaluate expression and induction of the vector promoter by doxycycline.
• VEGF expression will be evaluated on a per-cell basis following injection.
• FIG. 16 exemplifies a product retention study.
• MSC/VEGF cells were engineered to produce luciferase for imaging uses only. 100,000 cells per flank were injected and imaged over time. Mice were evaluated for the duration of retention. The study shows retention of at least 50% of the cells for the first week, but a log drop in cell number by day 14.
• FIG. 17 depicts the pCCLc-TRE-VEGF-PGK-rtTA-P2A-TK lentiviral vector.
• VEGF expression is under control of the tet-responsive promoter, so that the growth factor will only be produced from MSC in the recipient while they take doxycycline tablets (Month 1).
• the vector also encodes the well-characterized "suicide gene" TK, for additional biosafety. This gene allows eradication of all transduced cells if the antiviral ganciclovir is administered.
• FIG. 18 provides an overview of the MSC/VEGF process.
• MSCs mesenchymal stem cells
• VEGF vascular endothelial growth factor
• Marrow obtained from a qualified donor (Lonza) is plated and cultured under Good Manufacturing Practice (GMP) conditions following well-established Standard Operating Procedure (SOP) for MSC expansion at UC Davis.
• GMP Good Manufacturing Practice
• SOP Standard Operating Procedure
• the remainder of the cells are exposed to the lentiviral vector at a multiplicity of infection of approximately 20 (dependent on titer of the GMP vector lot), to generate the MSC/VEGF lot.
• This allows for insertion of 1-2 vector copies per cellular genome.
• the cellular product is expanded and tested extensively, then cryopreserved. Viability, sterility, freedom from endotoxin and mycoplasma, inserted vector copy numbers per genome and potency (VEGF production) are performed at each batch.
• the GMP grade transduction, banking and qualification of MSC/VEGF is controlled by GMP SOPs, and overseen by Quality Control and Quality Assurance. A certificate of analysis is issued after completion of these tests.
• the frozen product When needed for infusion, the frozen product is shipped, under controlled conditions, to the clinical site(s). There the product is thawed according to SOPs used for bone marrow stem cell transplantation, release tests are performed (gram stain and viability) and delivered to the surgical suite for intramuscular injection. In some studies the MSC/VEGF will be thawed and plated for 24-48 hours in hypoxic conditions prior to administration to the patient.
• FIG. 19 shows Human MSC beginning to grow out of a bony spicule obtained by gravity sedimentation from a normal donor bone marrow aspirate as described in Example 3 (day 3 after plating).
• FIG. 20 shows expanded human MSC at 75 % confluence as described in Example 3. This passage 3 culture was grown out of bone marrow spicules as described. The cells have a fairly uniform myofibroblastic appearance.
• FIG. 21 shows the phenotype (by FACS assay) of Expanded Marrow Stromal Cells/Mesenchymal Stem Cells (MSCs) from human bone marrow.
• the phenotype depicted is: CD34-/CD45-/CD105+/CD90+/CD73+.
• SEQ ID NO: 1 is the nucleotide sequence encoding for the 165 A VEGF isoform.
• SEQ ID NO: 2 is the nucleotide sequence of an example vector of the disclosure. The following features are in the vector at the indicated sequence location: 5601..6116 mPGK; 132..992 AMP (AmpiciUon resistance gene); 2358..3070 CMV enhanced 5'LTR"; 2892..2988 “R region 5'LTR”; 2990..3072 "U5 region 5'LTR”; 3120..3258 "PSI";
• a cell includes a plurality of cells, including mixtures thereof.
• compositions and methods include the recited elements, but not excluding others.
• Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like.
• Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention or process steps to produce a composition or achieve an intended result.
• isolated refers to molecules separated from other DNAs or R As, respectively that are present in the natural source of the macromolecule.
• isolated nucleic acid is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
• isolated is also used herein to refer to polypeptides, proteins and/or host cells that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.
• isolated means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature.
• an isolated cell is a cell that is separated form tissue or cells of dissimilar phenotype or genotype.
• polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof does not require “isolation” to distinguish it from its naturally occurring counterpart.
• the DNA viruses constitute classes I and II.
• the RNA viruses and retroviruses make up the remaining classes.
• Class III viruses have a double-stranded RNA genome.
• Class IV viruses have a positive single-stranded RNA genome, the genome itself acting as mRNA
• Class V viruses have a negative single-stranded RNA genome used as a template for mRNA synthesis.
• Class VI viruses have a positive single- stranded RNA genome but with a DNA intermediate not only in replication but also in mRNA synthesis.
• Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell.
• the integrated DNA form is called a provirus.
• polynucleotide refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof.
• Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown.
• polynucleotides a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
• a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
• modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
• the sequence of nucleotides can be interrupted by non-nucleotide components.
• a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
• the term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this invention that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
• a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is R A.
• A adenine
• C cytosine
• G guanine
• T thymine
• U uracil
• polynucleotide sequence is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
• Homology refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or “non-homologous" sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present invention.
• a polynucleotide or polynucleotide region has a certain percentage (for example, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.
• This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology.
• default parameters are used for alignment.
• One alignment program is BLAST, using default parameters.
• An equivalent nucleic acid, polynucleotide or oligonucleotide is one having at least 80 % sequence identity, or alternatively at least 85 % sequence identity, or alternatively at least 90 % sequence identity, or alternatively at least 92 % sequence identity, or alternatively at least 95 % sequence identity, or alternatively at least 97 % sequence identity, or alternatively at least 98 % sequence identity to the reference nucleic acid, polynucleotide, or oligonucleotide.
• amplification of polynucleotides includes methods such as PCR, ligation amplification (or ligase chain reaction, LCR) and amplification methods. These methods are known and widely practiced in the art. See, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 and Innis et al, 1990 (for PCR); and Wu et al. (1989) Genomics 4:560-569 (for LCR).
• the PCR procedure describes a method of gene amplification which is comprised of (i) sequence-specific hybridization of primers to specific genes within a DNA sample (or library), (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a DNA polymerase, and (iii) screening the PCR products for a band of the correct size.
• the primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e. each primer is specifically designed to be complementary to each strand of the genomic locus to be amplified.
• Primers useful to amplify sequences from a particular gene region are preferably complementary to, and hybridize specifically to sequences in the target region or its flanking regions. Nucleic acid sequences generated by amplification may be sequenced directly. Alternatively the amplified sequence(s) may be cloned prior to sequence analysis. A method for the direct cloning and sequence analysis of enzymatically amplified genomic segments is known in the art.
• a “gene” refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated.
• ORF open reading frame
• the term "express” refers to the production of a gene product.
• expression refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is
• polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
• a "gene product” or alternatively a “gene expression product” refers to the amino acid (e.g., peptide or polypeptide) generated when a gene is transcribed and translated.
• Under transcriptional control is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, depends on its being operatively linked to an element which contributes to the initiation of, or promotes, transcription. "Operatively linked” intends the polynucleotides are arranged in a manner that allows them to function in a cell. In one aspect, this invention provides promoters operatively linked to the downstream sequences, e.g., suicide gene, VEGF, 165A VEGF, tet activator, etc...
• encode refers to a polynucleotide which is said to "encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof.
• the antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
• a "probe” when used in the context of polynucleotide manipulation refers to an oligonucleotide that is provided as a reagent to detect a target potentially present in a sample of interest by hybridizing with the target.
• a probe will comprise a detectable label or a means by which a label can be attached, either before or subsequent to the hybridization reaction.
• a "probe” can be a biological compound such as a polypeptide, antibody, or fragments thereof that is capable of binding to the target potentially present in a sample of interest.
• Detectable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. Detectable labels can also be attached to a polynucleotide, polypeptide, antibody or composition described herein.
• a “primer” is a short polynucleotide, generally with a free 3' -OH group that binds to a target or "template” potentially present in a sample of interest by hybridizing with the target, and thereafter promoting polymerization of a polynucleotide
• PCR polymerase chain reaction
• a “polymerase chain reaction” is a reaction in which replicate copies are made of a target polynucleotide using a "pair of primers” or a “set of primers” consisting of an "upstream” and a “downstream” primer, and a catalyst of polymerization, such as a DNA polymerase, and typically a thermally-stable polymerase enzyme.
• Methods for PCR are well known in the art, and taught, for example in
• a primer can also be used as a probe in hybridization reactions, such as Southern or Northern blot analyses. Sambrook and Russell (2001), infra.
• Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
• the hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
• the complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.
• a hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
• Hybridization reactions can be performed under conditions of different
• stringency In general, a low stringency hybridization reaction is carried out at about 40 °C in 10 x SSC or a solution of equivalent ionic strength/temperature. A moderate stringency hybridization is typically performed at about 50 °C in 6 x SSC, and a high stringency hybridization reaction is generally performed at about 60 °C in 1 x SSC.
• Hybridization reactions can also be performed under "physiological conditions" which is well known to one of skill in the art.
• a non-limiting example of a physiological condition is well known to one of skill in the art.
• polynucleotides are described as "complementary”.
• a double-stranded polynucleotide can be “complementary” or “homologous” to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second.
• “Complementarity” or “homology” is quantifiable in terms of the proportion of bases in opposing strands that are expected to form hydrogen bonding with each other, according to generally accepted base-pairing rules.
• propagate or “expand” means to grow a cell or population of cells.
• growing also refers to the proliferation of cells in the presence of supporting media, nutrients, growth factors, support cells, or any chemical or biological compound necessary for obtaining the desired number of cells or cell type.
• the term "culturing” refers to the in vitro propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or
• vector refers to a non-chromosomal nucleic acid comprising an intact replicon such that the vector may be replicated when placed within a cell, for example by a process of transformation.
• Vectors may be viral or non-viral.
• Viral vectors include retroviruses, adenoviruses, herpesvirus, bacculoviruses, modified bacculoviruses, papovirus, or otherwise modified naturally occurring viruses.
• Exemplary non-viral vectors for delivering nucleic acid include naked DNA; DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles comprising DNA condensed with cationic polymers such as heterogeneous polylysine, defmed-length oligopeptides, and polyethylene imine, in some cases contained in liposomes; and the use of ternary complexes comprising a virus and polylysine-DNA.
• a "viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
• viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like.
• Alphavirus vectors such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying, et al. (1999) Nat. Med. 5(7):823-827.
• a vector construct refers to the polynucleotide comprising the lentiviral genome or part thereof, and a therapeutic gene.
• lentiviral mediated gene transfer or “lentiviral transduction” carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome.
• the virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell.
• Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell.
• the integrated DNA form is called a provirus.
• lentiviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.
• a "lentiviral vector" is a type of retroviral vector well-known in the art that has certain advantages in transducing nondividing cells as compared to other retroviral vectors. See, Trono D. (2002) Lentiviral vectors, New York: Spring- Verlag Berlin Heidelberg.
• Lentiviral vectors of this invention are based on or derived from
• oncoretroviruses the sub-group of retroviruses containing MLV
• lentiviruses the sub-group of retroviruses containing HIV
• ASLV, SNV and RSV all of which have been split into packaging and vector components for lentiviral vector particle production systems.
• the lentiviral vector particle according to the invention may be based on a genetically or otherwise (e.g. by specific choice of packaging cell system) altered version of a particular retrovirus.
• That the vector particle according to the invention is "based on" a particular retrovirus means that the vector is derived from that particular retrovirus.
• the genome of the vector particle comprises components from that retrovirus as a backbone.
• the vector particle contains essential vector components compatible with the RNA genome, including reverse transcription and integration systems. Usually these will include gag and pol proteins derived from the particular retrovirus.
• gag and pol proteins derived from the particular retrovirus.
• the majority of the structural components of the vector particle will normally be derived from that retrovirus, although they may have been altered genetically or otherwise so as to provide desired useful properties.
• certain structural components and in particular the env proteins may originate from a different virus.
• the vector host range and cell types infected or transduced can be altered by using different env genes in the vector particle production system to give the vector particle a different specificity.
• promoter refers to a region of DNA that initiates transcription of a particular gene.
• the promoter includes the core promoter, which is the minimal protion of the promoter required to properly initiate transcription and can also include regulatory elements such as transcription factor binding sites. The regulatory elements may promote transcription or inhibit transcription. Regulatory elements in the promoter can be binding sites for transcriptional activators or transcriptional repressors.
• a promoter can be constutive or inducible.
• a constitutive promoter refers to one that is always active and/or constantly directs transcription of a gene above a basal level of transcription.
• An inducible promoter is one which is capable of being induced by a molecule or a factor added to the cell or expressed in the cell.
• An inducible promoter may still produce a basal level of transcription in the absence of induction, but induction typically leads to significantly more production of the protein. Promoters can also be tissue specific. A tissue specific promoter allows for the production of a protein in a certain population of cells that have the appropriate transcriptional factors to activate the promoter.
• stem cell defines a cell with the ability to divide for indefinite periods in culture and give rise to specialized cells.
• stem cells are categorized as somatic (adult) or embryonic.
• a somatic stem cell is an undifferentiated cell found in a differentiated tissue that can renew itself (clonal) and (with certain limitations) differentiate to yield all the specialized cell types of the tissue from which it originated.
• An embryonic stem cell is a primitive (undifferentiated) cell from the embryo that has the potential to become a wide variety of specialized cell types.
• An embryonic stem cell is one that has been cultured under in vitro conditions that allow proliferation without differentiation for months to years.
• a clone is a line of cells that is genetically identical to the originating cell; in this case, a stem cell.
• Cell types that MSCs have been shown to differentiate into in vitro or in vivo include osteoblasts, chondrocytes, myocytes, and adipocytes.
• Mesenchyme is embryonic connective tissue that is derived from the mesoderm and that differentiates into hematopoietic and connective tissue, whereas MSCs do not differentiate into hematopoietic cells.
• Stromal cells are connective tissue cells that form the supportive structure in which the functional cells of the tissue reside.
• the MSC has the phenotype CD34-/CD45- /CD105+/CD90+/CD73+.
• an “antibody” includes whole antibodies and any antigen binding fragment or a single chain thereof.
• the term “antibody” includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule. Examples of such include, but are not limited to a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein, any of which can be incorporated into an antibody of the present invention.
• CDR complementarity determining region
• antibody is further intended to encompass digestion fragments, specified portions, derivatives and variants thereof, including antibody mimetics or comprising portions of antibodies that mimic the structure and/or function of an antibody or specified fragment or portion thereof, including single chain antibodies and fragments thereof.
• binding fragments encompassed within the term "antigen binding portion" of an antibody include a Fab fragment, a monovalent fragment consisting of the V L , V H , C L and CH, domains; a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the V H and C H , domains; a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, a dAb fragment (Ward et al.
• V L and V H are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv)).
• scFv single chain Fv
• Single chain antibodies are also intended to be encompassed within the term "fragment of an antibody.” Any of the above-noted antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for binding specificity and neutralization activity in the same manner as are intact antibodies.
• an “antibody” includes whole antibodies and any antigen binding fragment or a single chain thereof.
• the term “antibody” includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule. Examples of such include, but are not limited to a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein, any of which can be incorporated into an antibody of the present invention.
• CDR complementarity determining region
• antibody is further intended to encompass digestion fragments, specified portions, derivatives and variants thereof, including antibody mimetics or comprising portions of antibodies that mimic the structure and/or function of an antibody or specified fragment or portion thereof, including single chain antibodies and fragments thereof. It also includes in some aspects, antibody variants, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, antibody derivatives, a bispecific molecule, a multispecific molecule, a heterospecific molecule, heteroantibodies and human monoclonal antibodies.
• binding fragments encompassed within the term "antigen binding portion" of an antibody include a Fab fragment, a monovalent fragment consisting of the V L , V H , C L and CH, domains; a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the V H and C 3 ⁇ 4 domains; a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, a dAb fragment (Ward et al. (1989) Nature 341 : 544-546), which consists of a V H domain; and an isolated complementarity determining region (CDR).
• Fab fragment a monovalent fragment consisting of the V L , V H , C L and CH, domains
• F(ab')2 fragment a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region
• a Fd fragment consisting of
• V L and V H are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv)).
• scFv single chain Fv
• Single chain antibodies are also intended to be encompassed within the term "fragment of an antibody.” Any of the above -noted antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for binding specificity and neutralization activity in the same manner as are intact antibodies.
• antibody variant is intended to include antibodies produced in a species other than a mouse. It also includes antibodies containing post-translational modifications to the linear polypeptide sequence of the antibody or fragment. It further encompasses fully human antibodies.
• antibody derivative is intended to encompass molecules that bind an epitope as defined above and which are modifications or derivatives of a native monoclonal antibody of this invention.
• Derivatives include, but are not limited to, for example, bispecific, multispecific, heterospecific, trispecific, tetraspecific, multispecific antibodies, diabodies, chimeric, recombinant and humanized.
• bispecific molecule is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has two different binding specificities.
• multispecific molecule or “heterospecific molecule” is intended to include any agent, e.g. a protein, peptide, or protein or peptide complex, which has more than two different binding specificities.
• heteroantibodies refers to two or more antibodies, antibody binding fragments (e.g., Fab), derivatives thereof, or antigen binding regions linked together, at least two of which have different specificities.
• human antibody as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
• the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
• the term "human antibody” as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
• human antibody refers to an antibody in which substantially every part of the protein (e.g., CDR, framework, C L , C H domains (e.g., C H I, C H 2, Cm), hinge, (VL, VH)) is substantially non-immunogenic in humans, with only minor sequence changes or variations.
• antibodies designated primate monkey, baboon, chimpanzee, etc.
• rodent mouse, rat, rabbit, guinea pig, hamster, and the like
• other mammals designate such species, sub-genus, genus, sub-family, family specific antibodies.
• chimeric antibodies include any combination of the above.
• a human antibody is distinct from a chimeric or humanized antibody. It is pointed out that a human antibody can be produced by a non- human animal or prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and/or light chain) genes. Further, when a human antibody is a single chain antibody, it can comprise a linker peptide that is not found in native human antibodies.
• an Fv can comprise a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain.
• linker peptides are considered to be of human origin.
• a human antibody is "derived from” a particular germline sequence if the antibody is obtained from a system using human immunoglobulin sequences, e.g., by immunizing a transgenic mouse carrying human immunoglobulin genes or by screening a human immunoglobulin gene library.
• a human antibody that is "derived from” a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequence of human germline immunoglobulins.
• a selected human antibody typically is at least 90% identical in amino acids sequence to an amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as being human when compared to the germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences).
• a human antibody may be at least 95%, or even at least 96%>, 97%, 98%>, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene.
• a human antibody derived from a particular human germline sequence will display no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene.
• the human antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.
• the terms "monoclonal antibody” or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
• a "human monoclonal antibody” refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences.
• a population of cells intends a collection of more than one cell that is identical (clonal) or non-identical in phenotype and/or genotype.
• recombinant human antibody includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, antibodies isolated from a recombinant, combinatorial human antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences.
• Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences.
• such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
• composition is intended to mean a combination of active polypeptide, polynucleotide or antibody and another compound or composition, inert (e.g. a detectable label) or active (e.g. a gene delivery vehicle).
• a "pharmaceutical composition” is intended to include the combination of an active polypeptide, polynucleotide or antibody with a carrier, inert or active such as a solid support, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
• a pharmaceutical carrier encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
• the compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin (1975) Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton ).
• a "subject,” “individual” or “patient” is used interchangeably herein, and refers to a vertebrate, preferably a mammal, more preferably a human.
• Mammals include, but are not limited to, murines, rats, rabbit, simians, bovines, ovine, porcine, canines, feline, farm animals, sport animals, pets, equine, and primate, particularly human.
• the present invention is also useful for veterinary treatment of companion mammals, exotic animals and domesticated animals, including mammals, rodents, and the like which is susceptible to RNA and in particular, HIV viral infection.
• the mammals include horses, dogs, and cats.
• the human is an adolescent or infant under the age of eighteen years of age.
• “Host cell” refers not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
• Treating" or “treatment” of a disease includes: (1) preventing the disease, i.e., causing the clinical symptoms of the disease not to develop in a patient that may be predisposed to the disease but does not yet experience or display symptoms of the disease; (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms; or (3) relieving the disease, i.e., causing regression of the disease or its clinical symptoms.
• sensing refers to a patient or individual who has been diagnosed with or is predisposed to infection or a disease incident to infection.
• a patient may also be referred to being "at risk of suffering” from a disease because of active or latent infection. This patient has not yet developed characteristic disease pathology.
• an "effective amount” is an amount sufficient to effect beneficial or desired results.
• An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents of the present invention for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the subject, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. Treatment dosages generally may be titrated to optimize safety and efficacy. Typically, dosage-effect relationships from in vitro and/or in vivo tests initially can provide useful guidance on the proper doses for patient administration. In general, one will desire to administer an amount of the compound that is effective to achieve a serum level commensurate with the
• RNA virus replication is an amount sufficient to inhibit RNA virus replication ex vivo, in vitro or in vivo.
• administration shall include without limitation, administration by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implant), by inhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository) or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.) and can be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration.
• the invention is not limited by the route of administration, the formulation or dosing schedule.
• This invention provides a vector comprising, or alternatively consisting essentially of, or yet further consisting of: a nucleic acid encoding a 165 A isoform VEGF protein and a promoter that regulates expression of the nucleic acid encoding the VEGF.
• the backbone contains essential sequences for integration into a target cell genome.
• the term "vector” intends a recombinant vector that retains the ability to infect and transduce non-dividing and/or slowly-dividing cells and integrate into the target cell's genome.
• the vector is derived from or based on a wild- type virus.
• the vector is derived from or based on a wild-type lentivirus. Examples of such, include without limitation, human immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV), simian immunodeficiency virus (SIV) and feline immunodeficiency virus (FIV).
• retrovirus can be used as a basis for a vector backbone such murine leukemia virus (MLV).
• MLV murine leukemia virus
• a viral vector according to the invention need not be confined to the components of a particular virus.
• the viral vector may comprise components derived from two or more different viruses, and may also comprise synthetic components. Vector components can be manipulated to obtain desired characteristics, such as target cell specificity.
• the recombinant vectors of this invention are derived from primates and non- primates.
• primate lentiviruses include the human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SIV).
• the non-primate lentiviral group includes the prototype "slow virus” visna/maedi virus (VMV), as well as the related caprine arthritis- encephalitis virus (CAEV), equine infectious anaemia virus (EIAV) and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).
• U.S. Patent No. 6,924,123 discloses that certain retroviral sequence facilitate integration into the target cell genome.
• each retroviral genome comprises genes called gag, pol and env which code for virion proteins and enzymes. These genes are flanked at both ends by regions called long terminal repeats (LTRs).
• LTRs are responsible for proviral integration, and transcription. They also serve as enhancer-promoter sequences.
• the LTRs can control the expression of the viral genes. Encapsidation of the retroviral RNAs occurs by virtue of a psi sequence located at the 5' end of the viral genome.
• the LTRs themselves are identical sequences that can be divided into three elements, which are called U3, R and U5.
• U3 is derived from the sequence unique to the 3' end of the RNA.
• R is derived from a sequence repeated at both ends of the RNA
• U5 is derived from the sequence unique to the 5 'end of the RNA.
• the sizes of the three elements can vary considerably among different retroviruses.
• the site of poly (A) addition (termination) is at the boundary between R and U5 in the right hand side LTR.
• U3 contains most of the transcriptional control elements of the provirus, which include the promoter and multiple enhancer sequences responsive to cellular and in some cases, viral transcriptional activator proteins.
• gag encodes the internal structural protein of the virus.
• Gag protein is proteolytically processed into the mature proteins MA (matrix), CA (capsid) and NC (nucleocapsid).
• the pol gene encodes the reverse transcriptase (RT), which contains DNA polymerase, associated RNase H and integrase (IN), which mediate replication of the genome.
• RT reverse transcriptase
• I integrase
• the vector RNA genome is expressed from a DNA construct encoding it, in a host cell.
• the components of the particles not encoded by the vector genome are provided in trans by additional nucleic acid sequences (the "packaging system", which usually includes either or both of the gag/pol and env genes) expressed in the host cell.
• the set of sequences required for the production of the viral vector particles may be introduced into the host cell by transient transfection, or they may be integrated into the host cell genome, or they may be provided in a mixture of ways. The techniques involved are known to those skilled in the art.
• Viral vectors for use in this invention include, but are not limited to Invitrogen's pLenti series versions 4, 6, and 6.2 "ViraPower" system. Manufactured by Lentigen Corp.; pHIV-7-GFP, lab generated and used by the City of Hope Research Institute; "Lenti-X” lentiviral vector, pLVX, manufactured by Clontech; pLKO.l-puro,
• the vector is a viral vector.
• the viral vector is selected from the group consisting of a lentiviral vector, retroviral vector, adenovirus vector, adeno-associated virus vector, and alphavirus vector.
• the viral vector is a lentiviral vector.
• the vector comprises the polynucleotide of SEQ ID NO: 2.
• Non- viral vectors may include a plasmid that comprises a heterologous polynucleotide capable of being delivered to a target cell, either in vitro, in vivo or ex- vivo.
• the heterologous polynucleotide can comprise a sequence of interest and can be operably linked to one or more regulatory elements and may control the transcription of the nucleic acid sequence of interest.
• a vector need not be capable of replication in the ultimate target cell or subject.
• the term vector may include expression vector and cloning vector.
• the promoter that regulates expression of the nucleic acid encoding the VEGF protein is a constitutive promoter.
• the promoter is an inducible promoter.
• the promoter an inducible tetracycline promoter.
• Tet-Off and Tet-On Gene Expression Systems give researchers ready access to the regulated, high-level gene expression systems described by Gossen & Bujard (1992; Tet-Off) and Gossen et al. (1995; Tet-On). In the Tet-Off system, gene expression is turned on when tetracycline (Tc) or doxycycline (Dox; a Tc derivative) is removed from the culture medium.
• Tet-On In contrast, expression is turned on in the Tet-On system by the addition of Dox. Both systems permit gene expression to be tightly regulated in response to varying concentrations of Tc or Dox. Maximal expression levels in Tet systems are very high and compare favorably with the maximal levels obtainable from strong, constitutive mammalian promoters such as CMV (Yin et al., 1996). Unlike other inducible mammalian expression systems, gene regulation in the Tet Systems is highly specific, so interpretation of results is not complicated by pleiotropic effects or nonspecific induction. In E. coli, the Tet repressor protein (TetR) negatively regulates the genes of the tetracycline -resistance operon on the TnlO transposon.
• TetR Tet repressor protein
• TetR blocks transcription of these genes by binding to the tet operator sequences (tetO) in the absence of Tc.
• TetR and tetO provide the basis of regulation and induction for use in mammalian experimental systems.
• the regulatory protein is based on a "reverse" Tet repressor (rTetR) which was created by four amino acid changes in TetR (Hillen & Berens, 1994; Gossen et al, 1995).
• the resulting protein, rtTA (reverse tTA also refered to tetracycline activator protein), is encoded by the pTet-On regulator plasmid.
• This gene may be in a separate vector as the VEGF 165 A gene or encoded on the same gene.
• MSCs may first be made to express a stable cell line with the rtTA, and then the VEGF 165 A with a TRE promoter can be delivered by gene transfer or by viral infection to the MSC cell line.
• the rtTA and TRE-VEGF 165 A may be encoded on the same vector.
• the VEGF 165 A is under control of the tetracyc line-response element, or TRE. TREs can be made in different ways.
• the vector further comprises, or alternatively consists essentially of, or yet further consists of a nucleic acid encoding a tetracycline activator protein ; and a promoter that regulates expression of the tetracycline activator protein.
• inducible systems useful in vectors, isolated cells, viral packaging systems, and methods described herein include regulation by ecdysone, by estrogen, progesterone, chemical inducers of dimerization, and isopropyl-beta-Dl- thiogalactopyranoside (EPTG).
• Promoters useful in this disclosure can be constitutive or inducible.
• Some examples of promoters include SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter.
• the promoter that regulates expression of the tetracycline activator protein is a constitutive promoter.
• the promoter is an inducible promoter, a tissue specifc promoter, or a promoter that regulates expression temporally.
• the promoter is a phosphoglycerate kinase promoter (PGK).
• the vector further comprises, or alternatively consists essentially of, or yet further consists of a suicide gene and a promoter that regulates expression of the suicide gene.
• a suicide gene is one that allows for the negative selection of the cells.
• a suicide gene is used as a safety system, allowing the cells expressing the gene to be killed by introduction of a selective agent. This is desirable in case the recombinant gene causes a mutation leading to uncontrolled cell growth.
• TK herpes simplex virus thymidine kinase
• cytosine deaminase gene the varicella-zoster virus thymidine kinase gene
• nitroreductase gene the Escherichia coli gpt gene
• E. coli Deo gene also see, for example, Yazawa K, Fisher W E, Brunicardi F C: Current progress in suicide gene therapy for cancer. World J. Surg. 2002 July; 26(7):783-9.
• the suicide gene is the thymidine kinase (TK) gene. Cells expressing the TK protein can be killed using ganciclovir.
• nucleic acid encoding the tetracycline activator protein and the suicide gene are regulated by one promoter.
• Nucleic acids encoding different proteins may be regulated by the same promoter, produce one mRNA, and yet still result in the production of two different proteins. This can be accomplished in a variety of
• a protein cleavage site can be encoded in nucleic acids between the nucleic acids encoding for the protines.
• one mRNA is produced that encodes for both proteins and the protein cleavage site.
• the mRNA is translated into a chimeric polypeptide with both proteins linked by a protease cleavage site.
• the protein may then be cleaved by a protease that recognizes the cleavage site.
• the protease may be one that is endogenously expressed or exogenously expressed from a nucleic acid transferred into the cell by gene transfer methods.
• the vector further comprises, or alternatively consists essentially of, or yet further consists of a protease cleavage site between the suicide gene and the nucleic acid encoding the tetracycline activator protein.
• the protease cleavage site is the 2A protease cleavage site.
• An IRES or Internal Ribosome Entry Site may also be used to produce two proteins from the same promoter.
• nucleic acids encoding an IRES are cloned between the nucleic acids encoding each of the proteins.
• One mRNA is transcribed that encodes for both proteins, but the IRES site allows for separate translation of both proteins. Thus, two different proteins are produced by one mRNA.
• the nucleic acid encoding 165 A isoform VEGF comprises the polynucleotide of SEQ ID NO: 1, or a biological equivalent thereof.
• the biological equivalent of VEGF comprises a nucleic acid that hybridizes under conditions of high stringency to the complement of SEQ ID NO: 1 and encodes a VEGF protein.
• the biological equivalent thereof comprises a nucleic acid having at least 80 % sequence identity, or alternatively at least 85 % sequence identity, or alternatively at least 90 % sequence identity, or alternatively at least 92 % sequence identity, or alternatively at least 95 % sequence identity, or alternatively at least 97 % sequence identity, or alternatively at least 98 % sequence identity to SEQ ID NO: 1.
• the vector further comprises a marker or detectable label such as a gene encoding an enhanced green fluorescent protein (EGFP), red flouresence protein (RFP), green fluorescent protein (GFP) and yellow fluorescent protein (YFP) or the like.
• EGFP enhanced green fluorescent protein
• RFP red flouresence protein
• GFP green fluorescent protein
• YFP yellow fluorescent protein
• Genes may be delivered to the cell by a variety of mechanisms commonly known to those of skill in the art.
• Viral constructs can be delivered through the production of a virus in a suitable host cell. Virus is then harvested from the host cell and contacted with the target cell.
• Viral and non-viral vectors capable of expressing genes of interest can be delivered to a targeted cell via DNA/liposome complexes, micelles and targeted viral protein-DNA complexes. Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods of this invention.
• direct introduction of the proteins described herein to the cell or cell population can be done by the non-limiting technique of protein transfection, alternatively culturing conditions that can enhance the expression and/or promote the activity of the proteins of this invention are other non- limiting techniques.
• the host cells that are transfected with the vectors of this invention may include (but are not limited to) E. coli or other bacteria, yeast, fungi, insect cells (using, for example, baculoviral vectors for expression in SF9 insect cells), or cells derived from mice, humans, or other animals (e.g., mammals).
• E. coli or other bacteria yeast, fungi, insect cells (using, for example, baculoviral vectors for expression in SF9 insect cells), or cells derived from mice, humans, or other animals (e.g., mammals).
• In vitro expression of a protein, fusion, polypeptide fragment, or mutant encoded by cloned DNA may also be used.
• Those skilled in the art of molecular biology will understand that a wide variety of expression systems and purification systems may be used to produce recombinant proteins and fragments thereof.
• the invention also provides a viral packaging system comprising: the vector as described above, wherein the backbone is derived from a virus; a packaging plasmid; and an envelope plasmid.
• the packaging plasmid contains the nucleoside, capsid and matrix proteins. Examples of packaging plasmids are also described in the patent literature, e.g., U.S. Patent Nos. 7,262,049; 6,995,258; 7,252,991 and 5,710,037, incorporated herein by reference.
• the system also contains a plasmid encoding a pseudotyped envelope protein provided by an envelope plasmid.
• Pseudotyped viral vectors consist of vector particles bearing glycoproteins derived from other enveloped viruses or alternatively containing functional portions. See, for example U.S. Patent No. 7,262,049, incorporated herein by reference.
• the envelope plasmid encodes an envelope protein that does not cause the viral particle to unspecifically bind to a cell or population of cells.
• the specificity of the viral particle is conferred by the antibody binding domain that is inserted into the particle.
• suitable envelope proteins include, but are not limited to those containing the Staph, aureus ZZ domain.
• the choice of glycoprotein for use in the envelope is determined in part, by the antibody to which the particle may be conjugated.
• the packaging cell line is the HEK-293 cell line.
• suitable cell lines are known in the art, for example, described in the patent literature within U.S. Patent Nos. 7,070,994; 6,995,919; 6,475,786; 6,372,502; 6,365,150 and 5,591,624, each incorporated herein by reference.
• This invention further provides a method for producing a pseudotyped viral particle, comprising, or alternatively consisting essentially of, or yet further consisting of, transducing a packaging cell line with the viral system as described above, under conditions suitable to package the viral vector. Such conditions are known in the art and briefly described herein.
• the pseudotyped viral particle can be isolated from the cell supernatant, using methods known to those of skill in the art, e.g., centrifugation. Such isolated particles are further provided by this invention..
• This invention further provides the isolated pseudotyped viral particle produced by this method.
• the pseudotyped viral particle comprises, or alternatively consists essentially of, or yet further consists of a polynucleotide encoding a 165 A isoform VEGF protein and an envelope protein comprising a ZZ S. aureus domain.
• the isolated pseudotyped particles can be conjugate to one or more of an antibody or an antibody fragment (e.g. an fragment containing at least the Fc domain) that retains the ability to bind a pre-selected cell receptor.
• an antibody or an antibody fragment e.g. an fragment containing at least the Fc domain
• the antibodies are not species specific.
• the antibodies can be polyclonal or monoclonal and can be murine, ovine, human or other species.
• they can be chimeric or humanized.
• This invention also provides methods to prepare a pseudotyped viral particle by transducing a packaging cell line, as described herein with the vector, the envelope plasmid and the packaging plasmid under conditions that facilitate packaging of the vector into the envelope particle.
• the pseudotyped viral particle is a pseudotyped viral particle.
• the particles are separated from the cellular supernatant and conjugated to an antibody for cell-specific targeting.
• the complete vector particle is a viral, or alternatively a retroviral vector pseudotyped with a Sindbis virus glycoprotein envelope containing the ZZ domain of Protein A from Staphylococcus aureus.
• the genetic information of the viral vector particle is RNA which contains, on the 5' and 3' ends, the minimal LTR regions required for integration of the vector.
• the psi region which is required for packaging of the vector RNA into the particle. This region is followed by the RRE and cPPT sequences which enhance vector production by transporting the full length vector transcript out of the nucleus for efficient packaging into the vector particle.
• a nucleic acid encoding a suicide gene is encoded between the LTR regions.
• the full length RNA transcript is packaged inside the capsid of the vector particle which contains the nucleocapsid, capsid, and matrix proteins which are generated from the packaging plasmid delta-8.91.
• the reverse transcriptase polymerse which is generated from the packaging plasmid delta-8.91 is also located within the capsid with the RNA transcript.
• the capsid encases and protects the full length RNA transcript.
• Sindbis-ZZ glycoprotein envelope Surrounding the capsid/RNA complex is the Sindbis-ZZ glycoprotein envelope which is generated from the Sindbis-ZZ plasmid. This envelope, when conjugated with a specific monoclonal antibody, will direct the vector particle to specifically transduce a cell of interest that expresses a cell surface receptor recognized by the chosen monoclonal antibody.
• the vector particle is generated by a transient transfection protocol which includes a packaging cell line (HEK-293T cells), a lipofection reagent (Transit-293T, for example), and the three plasmids encoding the parts of the vector particle (delta-8.91 (packaging plasmid)), 165 A VEGF-containing vectors described herein (viral vector plasmid), and Sindbis-ZZ (envelope plasmid).
• HEK-293T cells packaging cell line
• a lipofection reagent Transit-293T, for example
• the three plasmids encoding the parts of the vector particle (delta-8.91 (packaging plasmid)
• 165 A VEGF-containing vectors described herein viral vector plasmid
• Sindbis-ZZ envelope plasmid
• HEK-293T cells are plated at 75% confluency in complete DMEM media 24 hours prior to transfection. After at least 24 hours post-plating of cells, the transfection mixture should be prepared. Three milliliters of serum free media is incubated with 150ul of the lipofection reagent for 20 minutes at room temperature. The three plasmids are then added to the media/lipofection reagent mixture at a ratio of 5:5:2 (packaging plasmid: viral vector plasmid: envelope plasmid) and incubated for 30 minutes. After this final incubation period, the media/lipofection reagent/DNA mixture is then added to the HEK-293T cells and left overnight for the transfection to occur. The next day, the transfection media is removed and fresh complete DMEM is added. Seventy-two hours later, the cell culture supernatant is collected and concentrated by ultracentrifugation at 20,000 rpm for 1.5 hours.
• the viral particle may be isolated from the supernantant.
• packaging cells can be transfected with the viral vector by known techniques of gene transfer, the cells can be incubated for a period of time to allow the virus to replicate.
• the viral particles may then be isolated from the cell culture medium or supernatant by aspirating the cell culture media from the cells.
• the virus particles may then be sterilized by techniques known in the art (filter sterilization, for example) and/or concentrated by techniques such as centrifugation and by using commercially available concentration reagents (available from, for example, Clontech, Cat. #631231).
• an isolated cell or population of cells comprising, or alternatively consisting essentially of, or yet further consisting of, viral particle of this invention.
• the isolated cell is a packaging cell line.
• an isolated cell or population of cells comprising, or alternatively consisting essentially of, or yet further consisting of, a nucleic acid encoding a 165 A isoform VEGF protein and an inducible promoter that regulates expression of the nucleic acid encoding the VEGF.
• the promoter is an inducible promoter as described herein.
• the isolated cell further comprises, or alternatively consists essentially of, or yet further consists of a nucleic acid encoding a tetracycline activator protein ; and a promoter that regulates expression of the tetracycline activator protein.
• the promoter that regulates expression of the tetracycline activator protein is a constitutive promoter.
• the promoter is a phosphoglycerate kinase promoter (PGK).
• the isolated cell further comprises, or alternatively consists essentially of, or yet further consists of a suicide gene and a promoter that regulates expression of the suicide gene.
• the suicide gene is the thymidine kinase gene.
• the nucleic acid encoding the tetracycline activator protein and the suicide gene are regulated by one promoter.
• the isolated cell further comprises, or alternatively consists essentially of, or yet further consists of a protease cleavage site between the suicide gene and the nucleic acid encoding the tetracycline activator protein.
• the protease cleavage site is the 2 A protease cleavage site.
• the isolalated cell comprises, or alternatively consists essentially of, or yet further consists of a nucleic acid comprising the polynucleotide of SEQ ID NO: 1, or a biological equivalent thereof.
• the biological equivalent of VEGF comprises a nucleic acid that hybridizes under conditions of high stringency to the complement of SEQ ID NO: 1 and encodes a VEGF protein.
• the biological equivalent thereof comprises a nucleic acid having at least 80 % sequence identity, or alternatively at least 85 % sequence identity, or alternatively at least 90 % sequence identity, or alternatively at least 92 % sequence identity, or alternatively at least 95 % sequence identity, or alternatively at least 97 % sequence identity, or alternatively at least 98 % sequence identity to SEQ ID NO: 1.
• the isolated cell comprises, or alternatively consists essentially of, or yet further, consists of the polynucleotide of SEQ ID NO: 2.
• the isolated cells described herein can be any of a cell of a species of the group of: murine, rats, rabbit, simians, bovines, ovine, porcine, canines, feline, farm animals, sport animals, pets, equine, and primate, and in particular a human cell.
• the cell is a stem cell.
• the isolated cell is a mesenchymal stem cell.
• the MSC has the phenotype CD34-/CD45- /CD105+/CD90+/CD73+.
• the isolated cell comprises a certain level of the 165A VEGF protein.
• the level of 165 A VEGF protein can be achieved by selecting an appropriate constitutive promoter that produces the desirable level of protein or by using an inducible system that regulates the amount of protein produced. These promoters and inducible systems have previously been described.
• the isolated cell comprises, or alternatively consists essentially of, or yet further, consists of at least about 5 xlO "6 ng of 165 A VEGF protein.
• the isolated cell comprises, or alternatively consists essentially of, or yet further, consists of at least about 1 xlO "7 ng, about 3 xlO "7 ng, about 5 xlO "7 ng, about 7 xlO "7 ng, about 9 xlO "7 ng, about 1 xlO "6 ng, about 2 xlO "6 ng, about 3 xlO "6 ng, about 4 xlO "6 ng, about 6 xlO "6 ng, about 7 xlO "6 ng, about 8 xlO "6 ng, about 9 xlO "6 ng, about 10 xlO "6 ng, about 12 xlO "6 ng, about 14 xlO "6 ng, about 16 xlO "6 ng, about 18 xlO "6 ng, about 20 xlO "6 ng, about 25 xlO "6 ng, about 30 xlO "
• compositions and Therapeutic Uses are Compositions and Therapeutic Uses
• compositions or kit comprising any one or more of the viral vectors, isolated cells, packaging system, pseudotyped viral, viral particle conjugated to an antibody or fragment thereof which in turn may optionally be conjugated to a cell and a carrier.
• the carrier is a pharmaceutically acceptable carrier.
• This disclosure also provides a method for treating peripheral artery disease or critical limb ischemia in a patient in need thereof comprising administering the isolated cell or an expanded population of the isolated cell as described herein.
• Critical limb ischemia is a severe blockage in the arteries of the lower extremities, which markedly reduces blood- flow. Patients with advanced CLI are at risk of leg amputation because blood flow to the tissue bed is occluded in atherosclerotic vessels.
• One approach to treatment of CLI is inducing formation of collateral blood vessels that bypass the primary blockage and restore tissue perfusion to initiate healing.
• Methods of this disclosure describe the use of 165 A VEGF -producing cells for promoting therapeutic angiogenesis. The MSC/VEGF cells migrate into hypoxic tissue to promote
• MSC/VEGF may be more active in promoting therapeutic angiogenesis in CLI than MSC therapies alone, VEGF protein administration, or plasmid-delivered VEGF administration.
• Another aspect relates to a method for promoting wound healing, promoting or increasing the rate of angiogenesis or wound healing, decreasing the size of a wound, or decreasing the time to wound healing in a patient in need thereof comprising
• a further aspect relates to a method for salvaging a limb in a patient with critical limb ischemia comprising administering the isolated cell or an expanded population of the isolated cell as described herein.
• This disclosure also relates to a method for increasing vascularization in a patient in need thereof comprising administering the isolated cell or an expanded population of the isolated cell as described herein.
• the patient has critical limb ischemia.
• the vascularization is increased in the ischemic limb.
• This therapy may also be useful for revascularizing other tissues such as the heart, kidney, and the brain after stroke.
• about 1-1000 million cells are administered to the patient in the methods described herein.
• This disclosure also provides isolated mesenchymal stem cells expressing the phenotype CD34-/CD45-/CD105+/CD90+/CD73+ and comprising a 165A VEGF polynucleotide and expanded populations of such cells. Also provided are composistions comprising the isolated cells or expanded populations of isolated cells described herein.
• a "composition” typically intends a combination of the active agent and another carrier, e.g., compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers.
• Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, terra-, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume.
• sugars including monosaccharides, di-, tri-, terra-, and oligosaccharides
• derivatized sugars such as alditols, aldonic acids, esterified sugars and the like
• polysaccharides or sugar polymers which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume.
• Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like.
• Representative amino acid/antibody components which can also function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like.
• Carbohydrate excipients are also intended within the scope of this invention, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.
• monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like
• disaccharides such as lactose, sucrose
• the term carrier further includes a buffer or a pH adjusting agent; typically, the buffer is a salt prepared from an organic acid or base.
• Representative buffers include organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine
• Additional carriers include polymeric
• excipients/additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-. quadrature. -cyclodextrin), polyethylene glycols, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as "TWEEN 20" and “TWEEN 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).
• excipients/additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-. quadrature. -cyclodextrin), polyethylene glycols, flavoring agents, antim
• the term "pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
• the compositions also can include stabilizers and preservatives and any of the above noted carriers with the additional provisio that they be acceptable for use in vivo.
• stabilizers and adjuvants see Martin REMINGTON'S PHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975) and Williams & Williams, (1995), and in the "PHYSICIAN'S DESK REFERENCE", 52 nd ed., Medical Economics, Montvale, N.J. (1998).
• the invention provides an article of manufacture, comprising packaging material and at least one vial comprising a solution of at least one agent or composition with the prescribed buffers and/or preservatives, optionally in an aqueous diluent, wherein said packaging material comprises a label that indicates that such solution can be held over a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36,40, 48, 54, 60, 66, 72 hours or greater.
• the invention further comprises an article of manufacture, comprising packaging material, a first vial comprising at least one agent or composition and a second vial comprising an aqueous diluent of prescribed buffer or preservative, wherein said packaging material comprises a label that instructs a patient to reconstitute the therapeutic in the aqueous diluent to form a solution that can be held over a period of twenty-four hours or greater.
• the formulations of the present invention can be prepared by a process which comprises mixing at least one agent or composition and a preservative selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben, (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal or mixtures thereof in an aqueous diluent.
• a preservative selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben, (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal or mixtures thereof in an aqueous d
• a measured amount of at least one antibody in buffered solution is combined with the desired preservative in a buffered solution in quantities sufficient to provide the antibody and preservative at the desired concentrations.
• Variations of this process would be recognized by one of skill in the art, e.g., the order the components are added, whether additional additives are used, the temperature and pH at which the formulation is prepared, are all factors that can be optimized for the concentration and means of administration used.
• compositions and formulations can be provided to patients as clear solutions or as dual vials comprising a vial of agent or composition that is reconstituted with a second vial containing the aqueous diluent.
• Either a single solution vial or dual vial requiring reconstitution can be reused multiple times and can suffice for a single or multiple cycles of patient treatment and thus provides a more convenient treatment regimen than currently available.
• Recognized devices comprising these single vial systems include pen-injector devices for delivery of a solution such as BD Pens, BD Autojectore, Humaject®, NovoPen®, B-D®Pen, AutoPen®, and OptiPen®,
• Methods of delivery include but are not limited to intra-arterial, intra-muscular, and intravenous.
• the compositions or cells are administed by intravenous injection.
• compositions or cells are administered by intramuscular injection.
• the compositions may be administered in one injection or in multiple injections.
• ischemic areas of the diseased limb may also be directly injected into ischemic areas of the diseased limb.
• Solutions containing the cells can be prepared in suitable diluents such as water, ethanol, glycerol, liquid polyethylene glycol(s), various oils, and/or mixtures thereof, and others known to those skilled in the art.
• suitable diluents such as water, ethanol, glycerol, liquid polyethylene glycol(s), various oils, and/or mixtures thereof, and others known to those skilled in the art.
• chlorobutanol ascorbic acid, parabens, thermerosal, or the like. It may also be preferable to include agents that alter the tonicity such as sugars or salts.
• EXAMPLE 1 Effects on proliferation and differentiation of multipotent bone marrow stromal cells engineered to express growth factors for combined cell and gene therapy
• MSCs mesenchymal stem cells/bone marrow stromal cells
• this over-expression may alter the fate of the MSCs.
• This example describes the effects of over-expressing the growth factors bFGF, PDGF- BB, TGF- ⁇ and VEGF in human bone marrow-derived MSCs. Ectopic expression of bFGF or PDGF-B lead to highly proliferating MSCs and lead to a robust increase in osteogenesis. In contrast, adipogenesis was strongly inhibited in MSCs over-expressing PDGF-B and only mildly affected in MSCs over-expressing bFGF.
• TGF- ⁇ blocked both osteogenic and adipogenic differentiation while inducing the formation of stress fibers and increasing the expression of the smooth muscle marker calponin-1 and the chondrogenic marker collagen type II.
• MSCs over- expressing VEGF did not vary from control MSCs in any parameters, likely due to the lack of VEGF receptor expression on MSCs.
• MSCs engineered to over-express VEGF strongly induced the migration of endothelial cells and enhanced blood flow restoration in a xenograft model of hind limb ischemia.
• MSCs multipotent mesenchymal stem cells/marrow stromal cells
• MSCs represent an ontologic and phylogenetic vestige of ancestors with regenerative potential, as found during early development of mammals or adult newts, salamanders and fishes.
• MSCs can be isolated from virtually all vascularized tissues and are proposed to correspond with the pericyte compartment.
• Bone marrow- derived MSCs can reconstitute bone and bone marrow stroma at ectopic sites in immunodeficient mice and have been used for various cell therapies to treat, among many others, graft vs. host disease, cardiac infarction and epidermal fistulas.
• MSCs are considered not to contribute significantly by direct differentiation and replacement of the damaged tissue, but rather to perform as trophic mediators, promoting tissue repair by production and release of soluble factors that inhibit inflammation, reduce fibrosis and induce
• the regeneration process requires orchestration of various signals including basic fibroblast growth factor (bFGF or FGF-2), platelet derived growth factor B (PDGF- BB), transforming growth factor betal (TGF- ⁇ ) and vascular endothelial growth factor (VEGF).
• bFGF or FGF-2 basic fibroblast growth factor
• PDGF- BB platelet derived growth factor B
• TGF- ⁇ transforming growth factor betal
• VEGF vascular endothelial growth factor
• Bone marrow aspirates from healthy human donors were purchased from Lonza (Allendale, NJ).
• bone marrow aspirates were passed through 90um pore strainers for isolation of bone spicules. Then, the strained bone marrow aspirates were diluted with equal volume of PBS and centrifuged over Ficoll (GE Healthcare, Waukesha, WI) for 30 minutes at 700 x g.
• Ficoll GE Healthcare, Waukesha, WI
• MSCs were transduced with third- generation lentiviral vectors with the form pCCLc-MNDU3-X-IRES-EGFP, where X is the insertion site for the full length cDNA of bFGF, PDGF-B, TGF- ⁇ , VEGF-A(165) or without insertion (as control).
• bFGF cDNA was subcloned from pBLAST45-hFGF2 (Invivogen, San Diego, CA), while cDNAs for PDGF-B, TGF- ⁇ and VEGF-A(165) were derived from pCMV-SPORT6 vectors (Open Biosystems, Huntsville, AL).
• MSCs were transduced with 2mg/ml protamine sulfate. The volume of lentivirus used for each transduction was determined by titration as the required volume to generate 80-95% GFP positive MSCs after 3 days.
• MSCs cultured in 6-well plates were transduced with the respective lentiviral vectors. 4 days later, medium was changed to 1 ml/well of MEM-alpha supplemented with 2% BSA and incubated for additional 24h. Then, supernatants were collected in order to confirm over- expression and secretion of each factor using a human angiogenesis array (cat# AAH- ANG-1-8), following manufacturer's instructions (RayBiotech, Inc. Norcross, GA).
• a human angiogenesis array catalog# AAH- ANG-1-8
• Cell proliferation Three days after transduction with the respective lentiviral vectors, 20,000 MSCs per well were plated in duplicate in 12-well plates, with a final concentration of 2,000 cells/cm . The day after and every second day, cells were detached by trypsinization treatment and counted with Trypan Blue exclusion dye using a hemocytometer.
• conditioned media of transduced cells was prepared by incubation of cells in 8ml MEM-alpha + 10% FBS/10 6 cells/75cm 2 flasks for 24h and stored at -80°C. Then, non- transduced MSCs (20,000 cells/cm") were incubated for 2h with the conditioned media that had been previously prepared, and proteins were immediately extracted using RIPA buffer (Pierce, Rockford, IL) supplemented with Halt protease and phosphatase inhibitor cocktail (Pierce). Proteins were loaded in 10% bis-acrylamide gels and transferred to nitrocellulose membranes. After blocking for lh, membranes were incubated with first antibodies overnight. Antibodies against phosphorylated and total Akt, and MAPK44/42 (ER l/2) were purchased from Cell Signaling Technology (Danvers, MA).
• RNA extraction and real time PCR Total RNA was extracted with RNA-Stat 60 (Iso-Test Diagnostics, Friendswood, TX), following manufacturer's instructions. Reverse transcription using 1 ⁇ g of RNA was performed using Taqman reverse transcription reagents (Applied Biosystems, Foster City, CA). For the semi-quantification of mRNA levels of bFGF and PDGF-B, a pre-made taqman primers/probe mix was used as provided by Applied Biosystems (accession numbers Hs00960934_ml and Hs00234042_ml respectively), using GAPDH as an internal control for these taqman assays (accession number Hs99999905_ml).
• SYBR Green Master mix (Applied Biosystems) was used for real time RT-PCR, using primers listed in Table 1. Primers were designed using OligoPerfectTM software (Invitrogen, Carlsbad, CA), unless denoted by a reference.
• Osteogenic differentiation For osteogenic induction assay, 10,000 MSC/cm were cultured for 14 days in osteogenic media (MEM-alpha + 10% FBS supplemented with 0.2mM ascorbic acid, 0.1 ⁇ dexamethasone, lOmM ⁇ -glycerolphosphate), with a medium change every 3-4 days. To measure alkaline phosphatase activity (ALP), at day 14 the cells were trypsinized and lysed for protein extraction, with 1.5mM Tris-HCl solution containing l .OmM ZnCl 2 , LOmM MgCl 2 and 1% Triton X- 100 for lOmin.
• ALP alkaline phosphatase activity
• Lysates were then centrifuged at 16,100 x g for 30min and incubated with p- nitrophenylphosphate liquid substrate solution (Sigma- Aldrich, St. Louis, MO) for 30min. Released p-nitrophenolate was determined spectrophotometrically at 405nm, while total protein concentration was determined with Coomassie staining (595nm). Calcium precipitation was measured based on a previously described protocol 26 . Briefly, cells were fixed with 10% v/v formalin solution for 15min, washed once with PBS, stained for 20min with gentle shaking with 1% w/v Alizarin Red S (ARS) indicator (Ricca).
• ARS Alizarin Red S
• Adipogenic differentiation MSCs were cultured in 6-well plates to confluence (approx. 15,000 cells/cm ) and cultured for 14 or 21 days, with medium change every 3-4 days, in adipogenic medium (MEM-alpha + 10% FBS supplemented with 0.5mM
• MSCs were trypsinized after 21 days in differentiation media and directly stained for 5min with 10 ⁇ g/ml Nile Red (MP Biomedicals, Illkirch, France) as originally described. Then, samples were washed once with PBS and measured by FACS at 580nm.
• HUVEC migration assay MSCs that had been engineered to over-express each GF were cultured in 6-well plates for 24h in standard media (10 5 cells/2ml/well). Then, supernatants were collected and tested for their effect on migration of human umbilical vein endothelial cells (HUVEC). For the migration assays, HUVEC were plated in 24- well plates (1.5xl0 5 cells/well) containing inserts from CytoSelect 24-well wound healing assay (Cell Biolabs Inc, San Diego, CA). After overnight incubation, inserts were removed creating a homogenous gap (or "scratch”) in the monolayer of cells.
• HUVEC human umbilical vein endothelial cells
• Hind Limb Ischemia model and blood flow restoration All rodent work was performed under an approved animal care protocol in the UC Davis Stem Cell Program immune deficient mouse core. Under anesthesia, NOD/SCID/p-2-microglobulin-deficient mice (Jackson Laboratories - West, Sacramento, CA) were subjected to unilateral hind limb ischemia surgeries as we have previously described. In brief, the mice were shaved and prepped, the right femoral artery and vein were exposed and dissected from the femoral nerve, and the proximal portion of the femoral artery was ligated with 6-0 braided silk sutures.
• Blood flow to the ischemic limb was measured immediately before cell transplantation (day 0) and again on day 4, 7, 14, 21, and 28 using a laser Doppler imager (Moor Instruments Ltd, Devon, UK) as previously described.
• a laser Doppler imager Moor Instruments Ltd, Devon, UK
• both the ischemic and the healthy legs were shaved and the animal was placed on a 37°C heating pad for 2-5 minutes before imaging to allow acclimation to the ambient conditions before blood flow to both legs was simultaneously measured.
• the blood flow to the ischemic leg is expressed as a ratio relative to the contralateral healthy leg.
• GF growth factors
• FIG. 1 A the mRNA of each of the four GFs was increased upon overexpression, although in different magnitudes.
• Each GF was also found to be increased on a protein level, as measured in supernatants of MSCs transduced with the respective GF, as shown using an angiogenesis array (FIG. IB) and ELISA (FIG. 1C).
• TGF-Pi induced an increase of bFGF at both the mRNA and protein levels.
• over-expression of PDGF-B lead to an over 100 -fold increase of PDGF-B at both mRNA and protein levels, while over-expression of the other growth factors remained within a linear range.
• the overexpression of these GFs lead to the activation of specific signaling pathways in MSCs (FIG. 1C). This was tested in non- transduced MSCs incubated for 1 hour in conditioned media collected from the GF over- expressing MSCs.
• Osteogenic differentiation of MSCs is increased by over-expression of bFGF and PDGF-B and inhibited by TGF- ⁇ : To determine the effect of over-expressing GF on the osteogenic differentiation potential of MSCs, transduced cells were cultured for 14 days in osteogenic media, then calcium precipitation, alkaline phosphatase activity and gene expression of osteogenic markers was measured. Calcium precipitation as determined by Alizarin Red S staining was enhanced upon over-expression of bFGF and PDGF-B, while over-expression of TGF- ⁇ strongly inhibited it (FIG. 3 A).
• ALP Alkaline phosphatase activity
• mRNA levels of the adipogenic markers peroxisome proliferator-activated receptor ⁇ (ppary) and fatty acid binding protein 4 (fabp4) were measured in transduced MSCs after 14 days in culture under adipogenic media. Consistently, over-expression of PDGF-B or TGF- ⁇ lead to reduced ppary and fabp4 mRNA levels, while enforced bFGF expression in MSCs lead to only a minor, but non-significant, effect (FIG. 4C and 4D).
• TGF- ⁇ - mediated effects on MSCs Ectopic expression of TGF- ⁇ in MSCs strongly inhibited their osteogenic and adipogenic differentiation potential (FIGs. 3 and 4). As mentioned above, parameters such as the alkaline phosphatase activity were found even below basal levels (i.e. MSCs incubated in normal culture media, FIG. 3B), suggesting that TGF- ⁇ does not block MSC differentiation to retain them in an immature, undifferentiated state, but rather induces the differentiation of MSCs to a different cell type.
• TGF- ⁇ directed strong morphological changes in MSCs that suggest that they are undergoing senescence; large, flattened polygonal shapes with actin bundles characteristic of stress fibers (FIG. 5A).
• MSCs over-expressing TGF- ⁇ did not appear to undergo true senescence, since the proliferation of cells was found to be normal.
• a spontaneous (i.e. in absence of a specific differentiation media) increase of the smooth muscle gene calponin-1 was observed (FIG. 5B) and the chondrogenic marker Col2A (FIG. 5C).
• control MSCs showed only a limited improvement of blood flow, while MSCs over-expressing VEGF showed a clear improvement in revascularization over time.
• TGF- ⁇ peptides associate with latent-TGF-P-binding proteins (LTBP) localizing to the extracellular matrix, therefore reducing active TGF- ⁇ levels in solution 33.
• LTBP latent-TGF-P-binding proteins
• supematants of MSCs that had been engineered to over-express TGF- ⁇ could not lead to activation of Smad2/3.
• Smad2/3 was found to be constitutively active in MSCs engineered to over-express TGF- ⁇ , possibly through the constant exposure to it.
• Significant effects on the biology and morphology of the MSCs engineered to express TGF- ⁇ were also noted, indicating that although the levels of protein were low, they were biologically active.
• TGF- ⁇ The effect of TGF- ⁇ on proliferation and differentiation has been shown to be cell type and concentration dependent. In this experimental setting, it was observed that over-expression of TGF- ⁇ did not significantly affect cell growth, but strongly inhibited both osteogenic and adipogenic differentiation. As previously described, TGF- ⁇ induces the formation of stress fibers in MSCs and increases the expression of smooth muscle markers (Narita et al, Cell Tissue Res 2008; 333(3): 449-59).
• TGF- ⁇ induces differentiation of MSCs into chondrocytes and is commonly used to prove the chondrogenic potential of MSCs in vitro (Heng BC, et al, Stem Cells 2004; 22(7): 1152-67).
• TGF- ⁇ induces differentiation of MSCs into chondrocytes and is commonly used to prove the chondrogenic potential of MSCs in vitro
• chondrocytes requires the growth of cells in a micromass pellet (Mackay AM, et al., Tissue Eng 1998; 4(4): 415-28).
• MSCs engineered to over-express TGF- ⁇ acquired a complex phenotype, characterized by the expression of some smooth muscle and chondrogenic-associated genes, but not others.
• VEGF vascular endothelial growth factor
• EXAMPLE 2 Intramuscular (IM) Injection of VEGF Producing MSC for the Treatment of Critical Limb Ischemia (CLI).
• IM Intramuscular
• CLI Critical Limb Ischemia
• MSC/VEGF is a sterile, cryopreserved suspension of cultured human
• mesenchymal stem cells transduced by a lentiviral vector to secrete human vascular endothelial growth factor 165 A (VEGF).
• VEGF vascular endothelial growth factor 165 A
• the product is formulated in physiological saline. In addition to meeting all safety and viability criteria, thawed product produces between 10-50 ng VEGF/ml/10 6 cells/24h in culture after dox induction, as measured by ELISA of culture supernatant.
• MSC/VEGF has been designed and will be administered to provide high local concentrations of vascular endothelial growth factor-Ai 6 5 (VEGF) in the affected limb of a CLI patient in order to induce revascularization, restore perfusion, and effect tissue repair.
• This product is to produce a gene modified cell therapy product (MSC/VEGF); safety modified, allogeneic donor-derived human mesenchymal stem cells (MSCs) engineered to increase amputation free survival of critical limb ischemia (CLI) patients.
• the goal is to achieve a statistically significant reduction in major amputation (limb salvage) in subjects with CLI (Rutherford category 4-5), as compared to a placebo control.
• Secondary endpoints may include: 1) wound healing from baseline to 6 months following the first treatment: Rate, size, and time to heal. 2) incidence of target limb revascularization (TLR). 3) time to major amputation of index leg or death. 4) change in baseline ABI/TBI. 5) change in baseline tissue perfusion measurements. Further endpoints may include improvement of rest pain, wound healing, or other clinically important parameters.
• VEGF levels in the bloodstream can be measured, the injection site is monitored for adverse events such as discoloration or angioma, and the tissue can be monitored for observable changes or palpatations.
• inverse PCR assays can be performed to rule out monoclonal expansion of transduced cells.
• Edema from overproduction of VEGF can be addressed by regulating the transgene with doxycycline. Persistent serious edema may necessitate adjusting dose of MSC/VEGF. Risks from using transduced cells, although low, include insertional mutagenesis.
• the vector has been modified with the TK suicide gene to permit destruction of cells if they were to show uncontrolled multiplication or VEGF secretion.
• product is administered one time, intramuscularly, as a split dose as described below.
• the final frozen MSC/VEGF product is thawed and immediately administered using angiosome mapping to guide multi-site IM injections to ischemic areas of the target limb. 50 to 150 million thawed cells per limb are administered.
• Patients can be treated serially with three increasing doses of MSC/VEGF (50 to 150 million cells).
• Angiosome mapping can be used to detect the most severely ischemic region of each patient's leg and to guide intramuscular delivery of MSC/VEGF into that region.
• Secondary endpoints include avoidance of major amputation (limb salvage) and improved tissue perfusion assessed by 7 methods, to indicate if MSC/VEGF has the anticipated biological activity in CLI patients within the dose range administered.
• VEGF expression is under inducible control by doxycycline administration, enabling control of VEGF made in the affected limb by administration of this antibiotic to patients, and the vector also has a thymidine kinase "suicide gene" for additional biosafety.
• the vector can be under GMP conditions and can be tested regularly for stability.
• MSC which are expanded from adult progenitor cells, are not prone to tumor formation, and appear to tolerize the immune response across donor mismatch. These attributes combine to allow MSC to reside in many different tissue types without disrupting the local micro environment, and in some cases, responding to the local environment with appropriate protein secretion.
• TK thymidine kinase
• suicide gene can be used to eliminate a graft if any adverse events occur.
• the most widely used suicide gene in human imaging and gene therapy trials is the HSV-thymidine kinase (TK) gene. It is the target of the FDA-approved drug ganciclovir that kills TK-expressing cells infected by Herpes virus.
• the clinical vector includes the TK gene. Efficient in vivo elimination of TK gene-transduced cells by administration of ganciclovir has been demonstrated with this strategy.
• MSC vascular endothelial growth factor
• bFGF or FGF-2 basic fibroblast growth factor
• PDGF-BB platelet derived growth factor B
• TGF- ⁇ transforming growth factor betal
• VEGF-A165 vascular endothelial growth factor
• MSC overexpress these growth factors.
• MSC were transduced with a lentiviral vector (FIG. 7), where the transgene corresponded to bFGF, PDGF-B, TGF- ⁇ or VEGF-Ai 65 .
• a lentiviral vector FIG. 7
• the transgene corresponded to bFGF, PDGF-B, TGF- ⁇ or VEGF-Ai 65 .
• As control MSC were transduced with the same lentiviral vector lacking this transgene.
• the MSC/VEGF stem cell product was chosen as the best candidate to study further in angiogenic assays, due to the lack of autocrine signaling of VEGF and the efficacy in inducing endothelial cell migration using the in vitro HUVEC potency assay (FIG. 9).
• the immune deficient mouse model of hindlimb ischemia (HLI), which is an established assay to detect angiogenic activity for cells to be used in revascularization therapies has previously been described in the art. In brief, a 1 cm segment of the right femoral artery is excised with collateral vessel ligation to induce complete hind limb ischemia.
• FIG. 10 shows the initial lack of blood flow through the major artery in the right hindlimb, and enhanced correction by IV infusion of a clinical development candidate, but not the control cell population.
• This model is an approved potency assay that provides a reproducible and clinically relevant method for assessing restoration of blood flow by human cells, in place under GLP conditions.
• the human stem cells can be tracked into the area of hypoxia using either luciferase/IVIS or fluorescent nanoparticle imaging (FIG. 11). This allows useful mechanistic studies for efficacy evaluations but neither tracking method can be used clinically. Previous data demonstrates the propensity of human stem cells to migrate toward areas of hypoxic damage.
• MSC/VEGF would also improve restoration of blood flow in mice after induction of unilateral hind limb ischemia. For this, 1 day after creating a hind limb ischemia in immune deficient mice, One million MSC transduced with either control or VEGF vectors were transplanted into the mice and blood flow on the ischemic limb was measured using laser Doppler imaging. As shown in FIG. 12 under these experimental conditions, control MSC showed only a limited improvement of blood flow, while MSC/VEGF showed a clear improvement in revascularization over time. This confirms the basic premise of the proposed study-that MSC/VEGF will be more effective than MSC in revascularizing ischemic tissue.
• the test article is safety-modified allogeneic donor bone marrow-derived human mesenchymal stem cells engineered to secrete Vascular Endothelial Growth Factor (MSC/VEGF), under the control of an inducible promoter.
• MSC/VEGF Vascular Endothelial Growth Factor
• Conditioned medium from MSC/VEGF is tested in vitro for efficacy in human endothelial cell wound closure and ELISA to measure the levels of active VEGF secreted, among others.
• the human MSC/VEGF development candidate was proven efficacious in a rodent model of hindlimb ischemia, through significant enhancement of revascularization as compared to non-gene-modified MSC (FIG. 12).
• the development candidate was proven efficacious in a rodent model of hindlimb ischemia, through significant enhancement of revascularization as compared to non-gene-modified MSC (FIG. 12).
• MSC/VEGF is safe as tested in sensitive long-term rodent models. No edema or other adverse events have been detected (FIG. 13).
• mice were tested six months after injection.
• MSC/VEGF were found to be safe, with no tumors or aberrant pathology observed in the tissues of transplanted mice, as assessed by GLP studies and a licensed pathologist.
• Four additional cohorts of NSG mice will be transplanted with the VEGF-Ai65-modified MSC to validate the safety of the gene-modified human MSCs. Tissue harvest will be performed and detailed biosafety assessments will be done using established techniques. Standard toxicology studies will be completed at the level of GLP with tissue analyses completed by board certified pathologists.
• GLP lots of allogeneic donor MSCs created using the GMP-grade vector are stable and effective in potency tests. No toxicity has been observed in high dose injections of MSC/VEGF. MSC transduced with other vectors for out to 18 months were tested in decade-long biosafety studies, and have not observed adverse events,
• the cell dose chosen for the toxicology studies represents 10 times the maximal dose to be administered in the human study, as corrected for animal weight. In the clinical trial the maximum cell dose that is administered to the patient is 15 x 10 7 cells. Since the average weight of an adult male patient is approximately 70kg, the
• mice are each analyzed under GLP conditions.
• Laser Doppler Imaging (LDPI) is used to quantitate the reduction in hind limb blood flow immediately following surgery. Recovery from ischemia is quantified by the ratio of blood flow in the ischemic vs. the contralateral (unligated) limb, as shown in FIG. 10.
• LDPI Laser Doppler Imaging
• Laser Doppler Perfusion Imaging (LDPI) is performed every three days to quantitate recovery of blood flow to ischemic limb over 4 weeks in transplanted mice versus sham-transplanted controls.
• the ratio of blood flow in the healthy vs. affected leg of each mouse is assessed, with standardized temperature, food, lighting, bedding, handling and other conditions between the four different groups of mice. This allows the best comparison from group to group under GLP conditions.
• mice are euthanized and tissues prepared for toxicology and cell retention assays.
• Vascularization of ischemic versus contralateral muscle is quantitated by capillary density using H+E with CD31 immunostaining (capillary density/mm ).
• Human cell recruitment to ischemic muscle is quantitated on slides by immunohistochemical analysis, as described.
• Tissues from each mouse are prepared for toxicology under GLP. All major organs are embedded in OCT medium for cryosectioning. Every 10th section (lOum thickness) is collected and stained with H&E. Stained slides are analyzed under light microscopy by a pathologist who is blinded to the treatment conditions. If any abnormal tissues are noted, fluorescence in situ hybridization (FISH) or polymerase chain reaction (PCR) is performed to determine whether the cells were of human or murine origin, as described in the art.
• FISH fluorescence in situ hybridization
• PCR polymerase chain reaction
• VEGF lentiviral vector supernatant has been generated. Scaled-up cultures of certified HEK-293T cells are transfected with a three plasmid lentiviral vector packaging system. 48 hours post-transfection, vector particles are collected and concentrated by spin filtration. After concentration, the GMP grade lentiviral vector is aliquoted and cryopreserved under GMP conditions. Aliquots are tested for sterility, endotoxin, mycoplasma, replication competent lentivirus and transducing titer using q- PCR. With this system, transducing titers in the high 10e9 to lOelO range are usually obtained.
• Lentiviral vector stored in the GMP facility can be tested for stability on a yearly basis.
• the viral titer obtained is at least about 10e4, 10e5, 10e6, 10e7, 10e8, 10e9, lOelO, lOel l, 10el2, 10el3, or 10el4.
• the viral lots are screened to ensure that they meet acceptable criteria for titer, endotoxin, sequence, sterility, and helper assay
• Donor testing is performed to the same standards as required for human bone marrow donors. Tests include Human Immunodeficiency Virus (HIV) 1 and 2, Hepatitis B, Hepatitis C, Cytomegalovirus (CMV), Syphilis, Human T-lymphotropic virus (HTLV) type 1 and type 2 and West Nile Virus. Qualified donors are negative for these human transmissible diseases. HLA typing is not required for human MSC transplantation since the cells are immune privileged.
• HLA Human Immunodeficiency Virus
• CMV Cytomegalovirus
• HTLV Human T-lymphotropic virus
• the clinical product is a human cellular product, and is an adult stem cell product.
• the clinical product is highly tested allogeneic human bone marrow-derived mesenchymal stem cells, transduced by the VEGF lentiviral vector (pCCLc-TRE-VEGF- PGK-rtTA-P2A-TK), expanded under strict adherence to Standard Operating Procedures (SOP) in place.
• SOP Standard Operating Procedures
• the process comprises 7 phases: 1. Receipt of donor bone marrow. 2. Direct plating and expansion of the adherent cell layer. 3. Split into MSC (10%) and
• MSC/VEGF (90%) lots at first passage. Transduction of MSC/VEGF lot. 4. Expansion of both lots, using spatial and temporal separation. 5. Harvesting of the expanded, adherent cell layer. 6. Qualification, labeling, freezing and storage of the transplantable, final products. 7. Generation of Certificates of Analysis (COA) for the final products.
• COA Certificates of Analysis
• the vectors and MSC are tested for sterility, endotoxin, mycoplasma, identity, sequence of transferred genes (DNA sequencing), and contaminating DNA (VSV-g DNA PCR). Acceptable lots meet sterility requirements of 14 days without an organism seen, LAL endotoxin level below acceptable limit, mycoplasma not detected by PCR, RCL not detected by HIV-1 p24 from transduction culture of susceptible cell lines and primary target cells, sequence of transferred gene identical to expected sequence, contaminating DNA within acceptable limits. Additionally, the MSC are also tested for viability by dye exclusion. Acceptable MSC, in addition to the guidelines listed above, exhibit viability greater than 70%.
• MSC lots are greater than 95% CD105+/73+/90+ and greater than 99% negative for CD45.
• Reagents include: Fetal Bovine Serum (heat inactivated @ 56°C for 30 mins.) screened and certified lot from FDA-approved vendor (BSE, virus, bacteria, and endotoxin free); Dulbecco's Modified Eagle Media (DMEM), store at 2-8° C, protect from light;
• Dulbecco's Phosphate Buffered Saline (without calcium or magnesium) (DPBS), store @ 15-30°C, Sterile for cell culture; Recombinant Trypsin (animal component- free); Trypan Blue, 0.5% in 100ml saline. Store at room temperature in small aliquot.; 100X (200mM) L- Glutamine or GlutaMAX store at 2-8 °C; Ficoll-Paque at room temperature; 70% Ethanol; and 10% Bleach.
• DPBS Dulbecco's Phosphate Buffered Saline (without calcium or magnesium)
• Supplies and equipment include: 15 ml sterile conical centrifuge tubes; 50 ml sterile conical centrifuge tubes; Serological pipettes, sterile, individually wrapped: 1 ml, 2 ml, 5 ml, 10 ml, 25 ml; 2 ml cryovials; 75 cm 2 (T 75) Tissue culture flasks with filter caps; 225 cm 2 (T 225) Tissue culture flasks with filter caps; 0.22 ⁇ Bottle-top filter flasks, 500 ml; 1 ml microcentrifuge tubes; Hemacytometer; 20 ⁇ Pipettor; Pipette tips for 20 ⁇ pipettor; Inverted Microscope; Centrifuge with GS-6R Rotor; 37°C, 5%> C0 2 Incubator.
• IMDM Iscove's Modified Dulbecco's Medium
• HI heat- inactivated
• HI Fetal calf serum* 5 ml L-glutamine (200 mM stock)
• 500 ⁇ hydrocortisone (10 " M stock).
• Marrow Filtration Screens Normal human bone marrow cells were obtained from screens used to filter marrow during harvest of allogeneic donors, (see Note-1); The cells from one harvest screen, from a normal donor, can be split between 4/T-75 vent-cap flasks, in 15 mis of stromal medium (see Materials) per flask. The cells are then expanded, as described below. Filter vent-cap flasks are used for long-term culture, (see Note-2),
• an optimal strategy is to use the mononuclear fraction, and also to recover the spicules from the bottom of the 50 ml ficoll tubes, since the small pieces of bone will fall through the density gradient.
• Expanding human MSC Plating MSC (initial seeding). To expand mesenchymal stem cells from total (RBC lysed) or ficolled marrow, cells prepared in DOM or D10HG are plated at a concentration of 5 x 10 6 mononuclear cells per ml in 75 cm 2 flasks, in 15-20 mis total volume at 37°C with 5% C0 2 . Spicules obtained from one harvest screen should be divided between four T-75 flasks containing 15 mis of DOM or D10HG each. Spicules obtained from the RBC pellet resulting from 10-15 mis of ficolled marrow can be plated in one T-75 flask, in 15 mis of medium. Cells are then expanded and transduced as described below.
• MSC expansion The MSC are allowed to adhere to the flasks overnight. The next morning, non-adherent cells can be gently flushed from the flasks and replated in a second flask, in the same medium. The initial flask is refed fresh medium. (DOM or D10HG) MSC colonies begin to develop as the cells expand out of the marrow spicules (FIG. 19). There are many other cells in the culture at this point. However, as the MSC grow and expand, the other cells differentiate out and/or can be removed. When the mesenchymal stem cells reach 70-80% confluency (70-80% of the plastic flask surface covered, FIG. 20), split them by trypsinization.
• mesenchymal stem cell population The cells will be rather “chunky”, not spindle shaped as will happen in straight FCS without the addition of horse serum.
• the cells can be collected using an EDTA-based cell dissociation buffer (rather than trypsin, which cleaves away many cell surface proteins), and then a FACS-based depletion can be done to remove CD45+ cells, including CD 14+ monocyte/macrophages, from the developing MSC monolayer. It is best to use trypsinization (trypsin-EDTA), to dissociate sub-confluent monolayers of primary mesenchymal stem cells from the flask. When the mesenchymal stem monolayer has the correct appearance (FIG.
• VSV-G pseudotyped lentiviral vector supernatant can be added once or twice at an MOI of 10-100, without the need for protamine sulfate.
• Transduced MSC are excellent vehicles from which to secrete proteins.
• the screens used to filter marrow during harvest are the richest source of mesenchymal stem cells. Many small bony spicules packed with stroma (as well as hematopoietic stem and progenitor cells) will get lodged in the screen, and can be easily removed by flushing. Filter vent-cap flasks are used for long-term culture, despite their greater cost as compared to standard screw-caps, because the risks for air-borne fungal spore contamination are high for cultures which can be grown for 1-2 months. The tightly closed, gas permeable filter vent caps reduce the risk of cross-contamination between flasks. It is advisable to perform at least one red cell lysis and wash before plating, if using this method. The washing technique is described in section 2.3. DOM is the richest medium and rapidly forces contaminating hematopoietic cells into erythroid and
• the spicules are a rich source of hematopoietic, as well as mesenchymal stem cells.
• DOM is the richest medium for MSC expansion without differentiation, and the horse serum rapidly forces contaminating hematopoietic cells into erythroid and monocytic differentiation, so that hematopoietic stem cells will not contaminate the stromal layer after 3 passages.
• a minimal medium, D10HG, which contains only fetal calf serum (Table IB) can also be used, but hematopoietic stem cells will survive horring on the stromal layer in this medium.
• MSC have not yet been expanded efficiently without the use of fetal calf serum, and it is imperative to screen the serum for optimal MSC growth without differentiation, when using either medium. In the fetal calf/horse serum mixture (DOM), the developing erythroid cells become nonadherent and are easily flushed away as the MSC layer develops and is expanded.
• a depletion step can be done at passage 2-3, to remove Glycophorin A+ cells.
• Early monocytes can also be removed by flushing, but mature macrophages are tightly adherent to the tissue culture flask and cannot be removed, even with trypsin. Therefore, the MSC can be taken to a new flask, while leaving the macrophages behind to be discarded.
• Mesenchymal stem cells will be readily trypsinized if they are in subconfluent monolayers. If they are allowed to become confluent, they form a 3 -dimensional tissue with excessive buildup of collagen and other extracellular matrix molecules between the layers of cells.
• the collagen layers are harder for the trypsin to digest than the adhesion foci with which the cells adhere to the plastic flask. The result is a useless sheet, or large chunks, of cells which will quickly deplete nutrients from the medium, and will necrose in the center. If the monolayer has numerous phase-bright macrophage contaminants, perform a CD45+ cell depletion using magnetic beads or FACS, or repeat steps 4-6. The resultant monolayer will be completely CD45 -negative, due to the loss of hematopoietic cells. No phase-bright cells will be seen adhering to the MSC monolayer (FIG. 20). It is imperative that the MSC not become confluent during the transduction procedure, but will remain in rapid growth.
• MSC contact inhibition in adherent cells, such as MSC, increases intracellular levels of the CDK inhibitor p27, which halts cell cycle.
• Target cells must traverse cell cycle to allow integration of retroviral vectors, and must be at least metabolically active for effective lentiviral vector transduction and integration.
• MSCs is not usually transduced or used for other studies until passage #3 or 4. At this point (FIG. 20), most hematopoietic cells will have been eliminated, except for mature macrophages which typically will comprise less than 1% of the culture.
• the cells also should be used for transduction, experiments, or transplantation between passage 3-6. By passage ten, they can begin to differentiate and become senescent.
• Protamine sulfate is a polycationic compound which neutralizes the negatively charged retroviral particles and cell surfaces. While the methodologies for transducing MSC are relatively simple— since in log phase the cells are rapidly dividing and incorporate vector very easily, in comparison to hematopoietic stem cells. The cells should be subconfluent when each aliquot of supernatant is added. Confluent cells are contact inhibited and will not divide to allow retroviral vector integration.
• MSC lentiviral vectors
• VSV-G envelope can cause cell fusion, resulting in multinucleate cells which appear overnight in the culture.
• MSC can and do "share" proteins with neighboring cells, through junction formation or other as-yet-unknown mechanisms. For this reason, Applicants recommend that MSC (marrow stromal cells) be plated at subconfluency for selective agents such as G418 to work effectively. This is also reflected in the fact that fluorescent markers such as eGFP can be shared between cells. Transduced cells dropped into a confluent plate of non-transduced MSC can cause a green "halo" to be seen in
• Expanded Marrow Stromal Cells/Mesenchymal Stem Cells (MSCs) from human bone marrow will have the phenotype CD34-/CD45-/CD105+/CD90+/CD73+, as shown in FIG. 21.

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