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  • C03B37/014—Manufacture of preforms for drawing fibres or filaments made entirely or partially by chemical means, e.g. vapour phase deposition of bulk porous glass either by outside vapour deposition [OVD], or by outside vapour phase oxidation [OVPO] or by vapour axial deposition [VAD]
  • C03B37/018—Manufacture of preforms for drawing fibres or filaments made entirely or partially by chemical means, e.g. vapour phase deposition of bulk porous glass either by outside vapour deposition [OVD], or by outside vapour phase oxidation [OVPO] or by vapour axial deposition [VAD] by glass deposition on a glass substrate, e.g. by inside-, modified-, plasma-, or plasma modified- chemical vapour deposition [ICVD, MCVD, PCVD, PMCVD], i.e. by thin layer coating on the inside or outside of a glass tube or on a glass rod
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  • C03B37/012—Manufacture of preforms for drawing fibres or filaments
  • C03B37/014—Manufacture of preforms for drawing fibres or filaments made entirely or partially by chemical means, e.g. vapour phase deposition of bulk porous glass either by outside vapour deposition [OVD], or by outside vapour phase oxidation [OVPO] or by vapour axial deposition [VAD]
  • C03B37/018—Manufacture of preforms for drawing fibres or filaments made entirely or partially by chemical means, e.g. vapour phase deposition of bulk porous glass either by outside vapour deposition [OVD], or by outside vapour phase oxidation [OVPO] or by vapour axial deposition [VAD] by glass deposition on a glass substrate, e.g. by inside-, modified-, plasma-, or plasma modified- chemical vapour deposition [ICVD, MCVD, PCVD, PMCVD], i.e. by thin layer coating on the inside or outside of a glass tube or on a glass rod
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  • C03B37/01—Manufacture of glass fibres or filaments
  • C03B37/012—Manufacture of preforms for drawing fibres or filaments
  • C03B37/014—Manufacture of preforms for drawing fibres or filaments made entirely or partially by chemical means, e.g. vapour phase deposition of bulk porous glass either by outside vapour deposition [OVD], or by outside vapour phase oxidation [OVPO] or by vapour axial deposition [VAD]
  • C03B37/018—Manufacture of preforms for drawing fibres or filaments made entirely or partially by chemical means, e.g. vapour phase deposition of bulk porous glass either by outside vapour deposition [OVD], or by outside vapour phase oxidation [OVPO] or by vapour axial deposition [VAD] by glass deposition on a glass substrate, e.g. by inside-, modified-, plasma-, or plasma modified- chemical vapour deposition [ICVD, MCVD, PCVD, PMCVD], i.e. by thin layer coating on the inside or outside of a glass tube or on a glass rod
  • C03B37/01884—Means for supporting, rotating and translating tubes or rods being formed, e.g. lathes
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  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
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  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10351—Methods of production or purification of viral material
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  • C12N2810/00—Vectors comprising a targeting moiety
  • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
  • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
  • C12N2810/6009—Vectors comprising as targeting moiety peptide derived from defined protein from viruses dsDNA viruses
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE

Definitions

• the use of recombinant adenovirus-de ved vectors according to one aspect of the present invention provides certain advantages for gene delivery.
• the Ad-derived vectors of the present invention possess all of the functional properties required for gene therapy including binding to epithelial cell receptors and penetration of endocytic vesicles.
• Therapeutic viral vectors of the present invention may also be engineered to target the receptors of and achieve penetration of non-epithelial cells; means of engineering viral vectors to accomplish these ends are described in detail herein below.
• the invention is also directed to an adenovirus vector packaging cell line comprising a stably integrated nucleic acid molecule as described above, an operatively-linked promoter and a nucleic acid sequence which encodes an adenovirus structural protein, wherein said TPL sequence consists essentially of a first TPL exon operatively linked to a complete second TPL exon operatively linked to a complete third TPL exon, wherein said packaging cell comprises an isolated nucleic acid molecule comprising an adenovirus tripartite leader (TPL) nucleotide, said TPL nucleotide sequence comprising (a) first and second different TPL exons or (b) first, second and third different TPL exons, said TPL exons selected from the group consisting of complete TPL exon 1 , partial TPL exon 1 , complete TPL exon 2 and complete TPL exon 3 and, wherein said isolated nucleic acid molecule may not comprise the nucleic acid sequence consisting of partial TPL exon
• This term is generally used herein to describe a plasmid vector that carries or delivers nucleotide sequences in or into a cell line (e.g., a packaging cell line) for the purpose of propagating therapeutic viral vectors of the present invention.
• a cell line e.g., a packaging cell line
• Expression or Delivery Vector Any plasmid or virus into which a foreign DNA may be inserted for expression in a suitable host cell - i.e., the protein or polypeptide encoded by the DNA is synthesized in the host cell's system.
• Vectors capable of directing the expression of DNA segments (genes) encoding one or more proteins are referred to herein as "expression vectors.” Also included are vectors which allow cloning of cDNA (complementary DNA) from mRNAs produced using reverse transcriptase.
• Isolated This term is used to indicate a nucleic acid or polypeptide sequence separated from the genetic environment from which the sequences were obtained. It may also mean altered from the natural state. For example, a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated", as the term is employed herein. Thus, a polypeptide or polynucleotide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention.
• isolated polypeptide or an “isolated polynucleotide” are polypeptides or polynucleotides that have been purified, partially or substantially, from a recombinant host cell or from a native source.
• a recombinantly produced version of a compounds can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31 -40 (1988). The terms isolated and purified are sometimes used interchangeably.
• a cell extract that contains the DNA or protein of interest should be understood to mean a homogenate preparation or cell-free preparation obtained from cells that express the protein or contain the DNA of interest.
• the term "cell extract” is intended to include culture media, especially spent culture media from which the cells have been removed.
• the viral vectors of the present invention may retain their ability to express the genome packaged within - i.e., they may retain their "infectivity" -- they do not act as infectious agents, however, to the extent that they cause disease in the subjects to whom they are administered for therapeutic purposes.
• WO 92/06693 (the disclosure of which is incorporated herein by reference).
• Other preferred therapeutic nucleotide sequences according to the present invention are capable of delivering HIV antisense oligonucleotides to latently-infected T cells via CD4.
• delivery of Epstein-Barr Virus (EBV) EBNa-1 antisense oligonucleotides to B cells via CR2 is capable of effecting therapeutic results.
• EBV Epstein-Barr Virus
• administration is often accomplished by first isolating a selected cell population from a patient such as lung epithelial cells, lymphocytes and the like followed by in vitro or ex vivo gene transfer of the therapeutic compositions of this invention and the replacement of the cells into the patient.
• In vivo therapy is also contemplated, e.g., via the administration of therapeutic compositions of this invention by various delivery means.
• aerosol administration and administration via subcutaneous, intravenous, intraperitoneal, intramuscular, ocular means and the like are also within the scope of the present invention.
• the invention also encompasses mammalian target cells infected with a preselected foreign nucleotide sequence produced by the methods disclosed herein.
• the target cells are selected from the group consisting of replicating, slow-replicating and non-replicating human cells.
• the dose of a biologic vector is somewhat complex and may be described in terms of the concentration (in plaque-forming units per milliliter (pfu/ml)), the total dose (in pfus), or the estimated number of particles administered per cell (the estimated multiplicity of infection or MOI).
• concentration in plaque-forming units per milliliter (pfu/ml)
• total dose in pfus
• MOI the estimated multiplicity of infection
• the therapeutic agent is a thymidine kinase metabolite such as ganciclovir (GCV), 6-methoxypurine arabinonucleoside (araM), or a functional equivalent thereof.
• GCV ganciclovir
• Both the thymidine kinase gene and the thymidine kinase substrate must be used concurrently in order to exert a toxic effect on the host cell.
• GCV is phosphorylated and becomes a potent inhibitor of DNA synthesis, whereas araM is converted to the cytotoxic anabolite araATP.
• the precise method of action or synergism is not relevant to therapeutic efficacy; what is relevant is the fact that the concurrent use of appropriate genes and therapeutic agents may effectively ameliorate a specific disease condition.
• the use of other anti-tumor genes provides a treatment for the uncontrolled cell growth or proliferation characteristic of tumors and malignancies.
• the present invention provides therapies to halt the uncontrolled cellular growth in a patient, thereby alleviating the symptoms or the disease or cachexia present in the patient.
• the effect of this treatment includes, but is not limited to, prolonged survival time of the patient, reduction in tumor mass or burden, apoptosis of tumor cells, or the reduction in the number of circulating tumor cells. Means of quantifying the beneficial effects of this therapy are well known to those of skill in the art.
• the present invention provides a recombinant adenovirus expression vector characterized by the partial or total deletion of one or more adenoviral structural protein genes, such as the gene encoding fiber, which allows the vector to accommodate a therapeutic, foreign nucleic acid sequence encoding a functional foreign polypeptide, protein, or biologically active fragment thereof.
• a therapeutic gene sequence may be introduced into a tumor mass by combining the adenoviral expression vector with a suitable pharmaceutically acceptable carrier. Introduction can be accomplished, for example, via direct injection of the recombinant Ad vector into the tumor mass.
• Example 3 The complete nucleotide sequence of pCLF is listed in SEQ ID NO: 8 where the nucleotide position 1 corresponds to approximately the middle of the pcDNA3 parent vector sequence.
• the 5' and 3 ends of the Ad5 fiber gene are located at respective nucleotide positions 1237-1239 with ATG and 2980-2982 with
• the pMAM backbone occupies nucleotide positions 1-1454 and 5006-11162 of SEQ ID NO: 12.
• the resulting plasmid containing E1 and fiber genes designated pE1/Fiber, provides both dexamethasone-inducible E1 function as described for DEX/E1 and expression of Ad ⁇ fiber protein as described above.
• a schematic plasmid map of pE1/Fiber, having 14466 bp, is shown in Figure 8.
• pDV44 fiber-deleted Ad5 genomic plasmid
• Figure 16A pDV44 contains a wild-type E4 region, but only the last 41 nucleotides of the fiber ORF (this sequence was retained to avoid affecting expression of the adjacent E4 transcription unit).
• Both pBHGI O and pDV44 contain unpackageable Ad5 genomes, and must be rescued by cotransfection and subsequent homologous recombination with DNA carrying functional packaging signals.
• Replacing or modifying the fiber gene in the vector chromosome would also require that the new fiber protein bind a receptor on the surface of the cells it which it is to be grown.
• the packaging cell approach will allow the generation of Ad particles containing a fiber which can no longer bind to its host cells, by a single round of growth in cells expressing the desired fiber gene. This will greatly expand the repertoire of fiber proteins which can be incorporated into particles, as well as simplifying the process of retargeting gene delivery vectors.
• the third TPL exon (exon 3) (nt 9644-9731 of the Ad ⁇ genome) was amplified from Ad ⁇ genomic DNA using the synthetic oligonucleotide primers ⁇ 'CTCAACAATTGTGGATCCGTACTCC3'(SEQ ID NO: 28) and ⁇ 'GTGCTCAGCAGATCTTGCGACTGTG3' (SEQ ID NO: 29).
• the resulting product was cloned to the BamHI and Bglll sites of p ⁇ E1 Sp1 a (Microbix Biosystems) using novel sites in the primers (shown in bold) to create plasmid pDV52.
• This plasmid contains a 1.2 kb BamHI/Bglll fragment consisting of the first TPL exon, the natural first intron, and the fused second and third TPL exons.
• the nucleotide sequence of the complete TPL containing the noted ⁇ ' and 3' restriction sites is shown in SEQ ID NO: 32 with the following nucleotide regions identified: 1-6 nt BamHI site; 7-47 nt first leader segment (exon 1); 48-1068 nt natural first intron (intron 1 ); 1069-1140 nt second leader segment (exon 2); 1141 -1146 nt fused BamHI and Bglll sites; 1147-1234 nt third leader segment (exon 3); and 1235-1240 nt Bglll site.
• E1-2a S8 cells were electroporated as previously described (Von Seggern et al., J. Gen Virol., 79: 1461 (1998)) with pDV61 , pDV67, or with pDV69, and stable lines were selected with zeocin (600 ⁇ g/ml).
• the cell line generated with pDV61 is designated 601.
• the cell line generated with pDV67 is designated 633 while that generated with pDV69 is designated 644.
• Candidate clones were evaluated by immunofluorescent staining with a polyclonal antibody raised against the Ad2 fiber. Lines expressing the highest level of fiber protein were further characterized.
• FIG. 20 The preparation of the cell lines and demonstration of stable nuclear expression of either wild-type Ad5 fiber protein or chimeric Ad ⁇ /Ad3 protein is shown in Figure 20.
• Figure 20 schematic diagrams are presented of the constructs used to generate the cell lines as well as immunofluorescence results indicating the presence of expressed fiber protein in the nucleus of the cells.
• An indirect immunofluorescence assay of A649 based cell lines which stably express the different Ad fibers is shown.
• Line 633 expresses the native Ad ⁇ fiber protein and line 644 expresses a chimeric fiber protein with the tail and shaft domains of the Ad ⁇ protein and the knob domain of the Ad3 fiber.
• Previous work (Stevenson et al., 1996) showed that a virus containing this protein had the tropism expected for Ad3.
• the plasmid pAdShuttleTK which contains the left part of the Ad chromosome with an RSV-driven TK gene inserted in place of the E1 region, is linearized by digestion with Notl.
• the nucleotide sequence of the pAdShuttleTK is shown in SEQ ID NO: 50.
• the large Clal fragment of Ad ⁇ . ⁇ gal. ⁇ F and the linearized pAdShuttleTK are cotransfected into 211 B cells, and an infectious adenovirus genome is generated by homologous recombination.
• a virus deleted for E1 , E3, and fiber that contains the TK cassette in the place of the E1 deletion is thus recovered.
• a virus containing any desired therapeutic gene of interest can be created in this manner by replacing the TK gene of the example with the gene of interest.
• a plasmid (pDV50) analogous to p ⁇ EI B ⁇ gal but containing a CMV-driven RDS gene was constructed as follows. First, a fragment containing the CMV promoter and enhancer was excised from pCHaMIEP by digestion with Hindlll, filling the overhanging ends with the large fragment of E. coli DNA polymerase 1 , ligation of BamHI linkers (5'CGCGGATCCCG3' SEQ ID NO: 61) to the blunt ends, and digesting with BamHI. The resulting fragment was then ligated into the BamHI site of p ⁇ E1sp1 a (Mikrobix) to create pDV4 ⁇ .
• a fragment containing the SV40 polyadenylation signal was amplified from pSV ⁇ gal (Promega) using the oligonucleotides ⁇ 'CTGACAAACTCAGATCTTGTTTATTG3' (SEQ ID NO: 51) and 5'GTCGACTCTAGAGGATCCAGA3' (SEQ ID NO: 62). This fragment was ligated into the Bglll site of pDV45 to create pDV46, using the unique BamHI and Bglll sites (bold type) in the primers.
• These particles have the capsid structure of adenovirus, and infect cells using the efficient fiber- and penton base-mediated pathway used by Ad.
• the hybrid genome is able to integrate into the cell's chromosomes by virtue of its AAV sequences.
• the AAV vector genome is inserted into the E1 region of a fiber-deleted vector, and the resulting vector is grown in packaging lines expressing either the Ad ⁇ or Ad37 fiber proteins.
• the particles recovered therefore have the tropisms expected from the respective fiber proteins combined with the ability to integrate their AAV genome into target cells.
• Such pseudotyping should be possible with any of a number of modified fiber proteins, as for the fiber-deleted vectors already described by us.

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