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WO1997012635A1 - Procede de traitement de la sclerose laterale amyotrophique - Google Patents

Procede de traitement de la sclerose laterale amyotrophique Download PDF

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Publication number
WO1997012635A1
WO1997012635A1 PCT/US1996/015824 US9615824W WO9712635A1 WO 1997012635 A1 WO1997012635 A1 WO 1997012635A1 US 9615824 W US9615824 W US 9615824W WO 9712635 A1 WO9712635 A1 WO 9712635A1
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WIPO (PCT)
Prior art keywords
cells
ofthe
cntf
hcntf
neurotrophic factor
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PCT/US1996/015824
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English (en)
Inventor
Patrick Aebischer
E. Edward Baetge
Joseph P. Hammang
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Cytotherapeutics, Inc.
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Application filed by Cytotherapeutics, Inc. filed Critical Cytotherapeutics, Inc.
Priority to AU72049/96A priority Critical patent/AU7204996A/en
Publication of WO1997012635A1 publication Critical patent/WO1997012635A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron

Definitions

  • This invention relates to treatment of amyotrophic lateral sclerosis (ALS) by delivery of hCNTF directly into the central nervous system, preferably using encapsulated hCNTF-secreting cells
  • Amyotrophic lateral sclerosis also known as Tooth, Lou Gehrig's disease or motor neuron disease, hereafter "ALS" is a progressive fatal disease of the voluntary motor system resulting in death within 2 to 5 years of onset The cause ofthe disease is unknown and no effective therapy has yet been devised Quantitative measurement ofthe isometric force generated by a set of muscles allows progression ofthe disease to be monitored
  • ALS is a common disease, with an annual incidence rate of 0 4 to 1 76 per 100,000 population (approximately 1,000-4,300 annually in the U S ) Most patients are more than 50 years old at the onset of symptoms and the incidence increases with each decade of life ALS occurs in a random pattern throughout the world, it is estimated that in about 5% of cases ALS is familial, being inherited as an autosomal dominant trait
  • ALS a chronic myelogenous obstructive senor
  • the pathology of ALS is characterized by vacuohzation and neurofiiament accumulation in the perikaryon and proximal axonal compartment
  • the process of neuronal injury appears to be implicated with oxidative stress and excitotoxic injury (Brown RHJ, "Amyotrophic Lateral Sclerosis recent insight from genetic and transgenic mice," Cell 80, pp 687- 692, (1995), Martinou J, Martinou I, Kato AC, "Cholinergic differentiation factor promotes survival of isolated rat embryonic motoneurons in vitro " Neuron 8; pp 737-744, (1992)
  • the cause of death is usually respiratory failure
  • Human ciliary neurotrophic factor is a potent neurotrophic factor which may have utility for the treatment of ALS Human CNTF and the gene encoding human CNTF are described in detail in United States patent numbers 4,997,929, 5,141,856 and co-pending United States patent application serial number 07/857,544 filed March 24, 1992 Each of those documents are specifically inco ⁇ orated herein by this reference Although the biological role of CNTF has not been conclusively established, CNTF appears to be released upon injury to the nervous system and may limit the extent of injury or neuronal damage
  • CNTF ciliary neurotrophic factor
  • CNS central nervous system
  • Regeneron's rhCNTF (ciliary neurotrophic factor) was shown to be toxic in a large Phase III (730 patients) ALS clinical trial At one and two months, there was a significant loss in muscle strength, significant weight loss and a loss of pulmonary function (Forced vital capacity or FVC) After two months, however, the differences diminished between drug and placebo, and by the end ofthe nine-month trial, there were no differences in the slope of isometric muscle strength loss (primary endpoint), FVC slope or weight loss slope Throughout the trial, there was never a difference in survival It is interesting to note that at two months, the number of neutralizing antibodies (antibodies against rhCNTF) significantly increased, possibly indicating that further damage from CNTF was prevented by these antibodies inactivating CNTF
  • Syntex/Synergen discloses methods for treating amyotrophic lateral sclerosis with CNTF That application specifies that the preferred mode of administration is subcutaneous or intravenous delivery at 0 5-50 ⁇ g/kg/day dosage In addition, the application notes that dosages of CNTF given to patients in clinical trials were roughly 2, 5, 10 and 20 ⁇ g/kg/day Another report suggests that daily doses of CNTF in the Syntex/Synergen trial were about 32, 130 and 325 ⁇ g per person Smith Barney Report July 7, 1995, at p 4 This would correlate to an actual dose of 0 5, 2 and 5 ⁇ g/kg/day (assuming an average weight of 65 kg/person)
  • a tethered device containing about 1 -3 x 10 6 genetically modified cells surrounded by a semipermeable membrane, is implanted intrathecally, it provides for slow, continuous release of hCNTF at a rate of 1 - 1500 ng day, preferably 250-1000 ng/day
  • the dosage should be sufficient to produce a measurable level of CNTF in the cerebrospinal fluid ("CSF")
  • CSF cerebrospinal fluid
  • CNTF central nervous system
  • CNTF central nervous system
  • CNTF central nervous system
  • the device and the cells it contains may be retrieved from the patient
  • Fig 7 Norris scores on patient 1 (Panel A), patient 2 (Panel B), patient 3 (Panel C), patient 4 (Panel D), and patient 5 (Panel E)
  • Fig 8 Plasmid map ofRP 3224E2, the pNUT vector containing the hCNTF gene and containing the TK gene at the NotI site
  • Figure 9 pNUT-hCNTF-NT expression vector
  • the hCNTF gene was subcloned into the DHFR-based pNUT expression vector MT-1 promoter mouse metallothionein I promoter, Ig signal sequence mouse immunoglobulin signal sequence, hGH-3'UT 3' untranslated human growth hormone region containing the polyadenylation signal; HSV-tk gene: he ⁇ es simplex virus thymidine kinase gene; NEO R gene neomycin resistance gene; SV40 promoter simian virus 40 promoter.
  • This invention relates to delivery of CNTF directly into the CNS without having to cross the blood brain barrier
  • the dosage provided is sufficient to achieve a measureable level of up to 1000 ng/ml in the cerebrospinal fluid (CSF), preferably between 0.01-1000 ng/ml in the CSF, most preferably between 0 1-100 ng/ml in the CSF
  • living cells are encapsulated in one or more semipermeable polymer capsules and surgically inserted (under local anesthesia) into the CNS
  • this invention may be useful in the treatment of ALS Sporadic ALS is the most common form ofthe disease and there are several important subtypes.
  • Classical ALS the most common subtype, is distinguished by both upper and lower motor neuron degeneration Progressive bulbar palsy (PBP) affects approximately 25% ofthe ALS population and in early stages involves atrophy of muscles innervated by neurons in the lower cranial brainstem, thereby affecting speech and mastication in its early stages
  • PMA progressive muscular atrophy
  • PMA progressive muscular atrophy
  • PLS primary lateral sclerosis
  • the rarest ofthe subfamilies targets the upper motor neurons
  • Other types ofthe disease include familial ALS, Western Pacific ALS, Juvenile ALS and ALS-like diseases with definable causes (e g , Post-polio syndrome, heavy metal intoxication, etc ) This technique provides several advantages over other delivery routes
  • Drug can be delivered to the CNS directly, which will reduce unwanted peripheral side effects,
  • Very small doses of drug can be delivered compared with subcutaneous injections, also leading to fewer side effects,
  • CNTF Due to its instability, CNTF cannot be easily administered continuously — potentially the best mode of therapy When administered outside of the CNS, via pump, over 90% of CNTF activity is lost within 12 hours It is also hypothesized that sustained delivery of CNTF may prevent the down-regulation of the CNTF receptor that typically occurs with repeated bolus administrations
  • CNTF is toxic when given in a bolus injection intrathecally (personal communication from Dr M Sendtner)
  • Large doses of CNTF can therefore not be delivered intrathecally, although intrathecal delivery may be more efficacious since it can act directly on the cell body receptors ofthe lower motoneurons (spinal motoneurons) and potentially on the upper motoneurons (cortical motoneurons, Betz cells) as well The latter are inaccessible by systemic delivery since the blood brain barrier inhibits the diffusion of CNTF directly into the central nervous system
  • CNTFs used in the present invention are the naturally-occurring human proteins, such as those described in U S patents 5,01 1,914, 5, 141,856; and 4,997,929 all of which are inco ⁇ orated herein by reference
  • Modified, truncated and mutein forms of CNTF are well known See, e.g., WO 94/09134 and WO 93/10233.
  • CNTF all of these forms of CNTF are contemplated by this invention.
  • active fragments of CNTF i.e., those fragments of CNTF having biological activity sufficient to achieve a therapeutic effect
  • present invention also encompasses both microbially produced and mammalian cell produced forms of CNTF
  • PEG polyethylene glycol
  • nucleic acid sequences ofthe genes encoding human and animal CNTFs and the amino acid sequences of such proteins are known See, e.g , U S patent numbers 4,997,929 and 5, 141,856, incorporated herein by reference
  • full length recombinant human CNTF (rhCNTF) is used
  • a gene of interest i e , a gene that encodes a suitable biologically active molecule, e g., CNTF
  • a suitable biologically active molecule e g., CNTF
  • the expression vector containing the gene of interest may then be used to transfect the desired cell line Standard transfection techniques such as calcium phosphate co-precipitation, DEAE-dextran transfection or electroporation may be utilized.
  • Standard transfection techniques such as calcium phosphate co-precipitation, DEAE-dextran transfection or electroporation may be utilized.
  • mammalian transfection kits may be purchased from e.g., Stratagene
  • a wide variety of host/expression vector combinations may be used to express the gene encoding CNTF, or other biologically active molecule of interest
  • Suitable promoters include, for example, the early and late promoters of SV40 or adenovirus and other known non-retroviral promoters capable of controlling gene expression
  • Useful expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as various known derivatives of SV40 and known bacterial plasmids, e g , pUC, pBlueScriptTM plasmids from E. coli including pBR322, pCRl, pMB9, pUC, pBlueScriptTM and their derivatives
  • a biologic gene of interest e.g., NGF
  • G418 resistance gene codes for aminoglycoside phosphotransferase (APH) which enzymatically inactivates G418 (100-500 ⁇ g/1) added to the culture medium Only those cells expressing the APH gene will survive drug selection usually resulting in the expression ofthe second biologic gene as well
  • APH aminoglycoside phosphotransferase
  • HSH hygromycin B phosphotransferase
  • promoters can be employed to direct the expression ofthe genes for G418 and hygromycin B and/or the biologic gene of interest
  • These promoters include, but are not limited to, the promoters of hDBH (human dopamine beta hydoxylase) (Mercer et al , Neuron. 7, pp 703-716, (1991)), hTH (human tyrosine hydroxylase) (Kaneda, et al., Neuron. 6, pp 583-594 (1991)), hPNMT (human phenylethanolamine N-methyltransferase) (Baetge et al , PNAS.
  • hDBH human dopamine beta hydoxylase
  • hTH human tyrosine hydroxylase
  • hPNMT human phenylethanolamine N-methyltransferase
  • mGFAP mouse glial fibrillary acidic protein
  • MBP myelin basic protein
  • mNF-L mouse neurofilament-light subunit
  • hPo human P 0 , the promoter for the gene encoding the major myelin glycoprotein in the peripheral nervous system
  • mMT rat neuron-specific enolase
  • expression vectors examples include the commercially available pRC/CMV, pRC/RSV, and pCDNAlNEO (InVitrogen)
  • the viral promoter regions directing the transcription of the drug selection and biologic genes of interest are replaced with one ofthe above promoter sequences that are not subject to the down regulation experienced by viral promoters within the CNS
  • the GFAP promoter would be employed for the transfection of astrocytes and astrocyte cell lines
  • the TH promoter would be used in PC 12 cells
  • the MBP promoter would be used in oligodendrocytes
  • the pNUT expression vector is used Baetge et al , PNAS.
  • the pNUT expression vector can be modified such that the DHFR coding sequence is replaced by the coding sequence for G418 or hygromycin drug resistance
  • the SV40 promoter within the pNUT expression vector can also be replaced with any suitable constitutively expressed mammalian promoter, such as those discussed above
  • the human CNTF gene was expressed from the above mentioned dihydrofolate reductase (DHFR)-based selection vector designated pNUT, which contains the cDNA ofthe mutant DHFR and the entire pUC18 sequence including the polylinker (Aebischer, P , et al , "Transplantation in humans of encapsulated xenogeneic cells without immunosuppression A preliminary report," Transplantation.
  • DHFR dihydrofolate reductase
  • the DHFR transcription unit is driven by the SV40 promoter and fused at its 3' end with the hepatitis B virus gene polyadenylation signal (approximately 200 bp 3' untranslated region) to ensure efficient polyadenylation and maturation signals
  • the hCNTF gene was expressed behind the mouse metallothionei I promoter and utilizes the human growth hormone polyadenylation signal sequence for 3' processing
  • the pNUT vector containing the CNTF gene insert was named RP3224D
  • This invention also contemplates use of a "suicide" gene in the transformed cells Most preferably, the cell carries the TK (thymidine kinase) gene as a safety measure, permitting the host cell to be killed in vivo by treatment with ganciclovir
  • a "suicide” gene is known in the art See, e g , Anderson, published PCT application WO 93/10218, Hamre, published PCT application WO 93/02556
  • the recipient's own immune system provides a first level of protection from adverse reactions to the implanted cells If encapsulated, the polymer capsule itself may be immuno-isolatory
  • the presence ofthe TK gene (or other suicide gene) in the expression construct adds an additional level of safety to the recipient ofthe implanted cells
  • the He ⁇ es Simplex Virus Thymidine kinase (HSV-TK) gene was inserted into the CNTF pNUT RP3224D construction This suicide gene was included to allow elimination ofthe transfected cells upon ganciclovir administration
  • the 1800 bp fragment was inserted into the NotI site ofthe pNUT vector, yielding RP 3224E2 Figure 8
  • a typical dosage of ganciclovir required to "kill" the cells is approximately 5 mg/kg
  • BHK cells baby hamster kidney (BHK) cells
  • BHK cells offer several advantages including unlimited availability, the possibility of rapid screening in vitro for the presence of pathogens from which cell banks are established, and the suitability for stable gene transfer using non-viral-based recombinant DNA techniques
  • Both allogeneic and xenogeneic cells may be used Use of a cell line of xenogeneic origin provides an additional advantage since the transplanted cells will be rejected by the host immune system in the event of device breakage These cells are surrounded with a permselective membrane which permits the diffusion of small molecules such as nutrients and trophic factors into and out ofthe polymer envelope, while excluding larger molecules ofthe immune system (antibodies, complement, etc ) While BHK cells are one preferred cell, a wide variety of cells may be used These include well known, publicly available immortalized cell lines as well as dividing primary cell cultures.
  • Suitable publicly available cell lines include, Chinese hamster ovary (CHO), mouse fibroblast (L-M), NIH Swiss mouse embryo (NIH/3T3), African green monkey cell lines (including COS-1, COS-7, BSC- 1 , BSC-40, BMT- 10 and Vero), rat adrenal pheochromocytoma (PC 12 and PC12A), AT3, rat glial tumor (C6), astrocytes and other fibroblast cell lines
  • Primary cells that may be used include, EGF-responsive neurospheres, bFGF-responsive neural progenitor stem cells derived from the CNS of mammals (Richards et al., PNAS 89, pp.
  • C 2 C, 2 cells are preferred
  • the cell types that can be employed for encapsulated cell therapy within the scope of this invention include cells from allogeneic and xenogeneic sources.
  • cells from allogeneic and xenogeneic sources One ofthe principal advantages of our encapsulated approach rests with the immunoisolatory properties ofthe membranes of this invention, and their ability to support cells that otherwise would not be appropriate for transplantation (i e , non-human sources, immortalized and/or tumor cell lines)
  • a particular advantage to using xenogeneic over allogeneic cells is that in the unlikely event of membrane failure, the xenogeneic cells are more likely to be targeted for destruction by the immune system when compared to allogeneic cells Furthermore, xenogeneic sources are easy to obtain and their use precludes the necessity for the use of human tissue which is difficult to obtain and fraught with societal and ethical considerations In addition, human tissue may contain adventitious agents that are more readily transmitted to the transplantation recipient Finally, use of xenogeneic tissue and cell lines for transplantation in humans removes the risks associated with the handling and processing of human tissue
  • Increased expression can be achieved by increasing or amplifying the copy number ofthe transgene encoding CNTF or other suitable biologically active molecule(s), using amplification methods well known in the art
  • amplification methods include, e.g., DHFR amplification (see, e.g , Kaufman et al , United States patent 4,470,461) or glutamine synthetase ("GS”) amplification (see, e.g , United States patent 5,122,464, and European published application EP 338,841)
  • rhCNTF is delivered intrathecally using encapsulated cells.
  • Baby hamster kidney (BHK) cells are genetically engineered to stably express and release recombinant human CNTF into the central nervous system (CNS).
  • the CNTF-pNUT expression vector is transfected into baby hamster kidney (BHK) cells using a standard calcium phosphate transfection procedure and selected with increasing concentrations of methotrexate ( 1 to 200 ⁇ M) over 8 weeks to produce stable amplified cell lines Following this selection, the engineered BHK cells were maintained in vitro in 50-200 ⁇ M methotrexate
  • the genetically altered cells are autologous to the host and may not require encapsulation
  • other molecules may be co-delivered to the CNS including neural growth factors, cytokines, hormones, and neurotransmitters (including peptide neurotransmitters)
  • neurotransmitters including peptide neurotransmitters
  • co-delivery of IGF-1, NGF, GDNF, BDNF, NT-3, NT-4/5, NT-6, CT-1, IFN- ⁇ and IFN- ⁇ are contemplated
  • CNTF and GDNF are co-delivered
  • CNTF and NT-4/5 are co-delivered
  • CNTF and BDNF are co-delivered.
  • CNTF and IGF-I are co-delivered
  • CNTF co-delivery of CNTF, GDNF and NT-4/5
  • a sixth embodiment there is co-delivery of CNTF, BDNF and NT-4/5
  • a seventh embodiment there is co-delivery of CNTF, BDNF and GDNF It will be appreciated that any combination ofthe foregoing molecules may be co-delivered with CNTF
  • the co-delivered molecules are delivered in a dose similar to that of CNTF (i.e., sufficient to achieve a measureable level up to 1000 ng/ml in the CSF, preferably between 0.01 - 1000 ng/ml in the CSF, most preferably between 0 1 ng/ml and 100 ng/ml)
  • Co-delivery can be accomplished in a number of ways Cells may be transfected with separate constructs containing the genes encoding the described molecules. Alternatively, cells may be transfected with a single construct containing two or more genes.
  • IVS internal ribosome entry sites
  • WO 94/24870 refers to use of more than two IRES for translation initiation from a single transcript, as well as to use of multiple copies of the same IRES in a single construct
  • Various modified RES sequences are known See, e.g., Mountford and Smith, Trends Genet.. 1 1, pp 179-84 (1995), Dirks et al., Gene. 128, pp 247-49 (1993), Martinez-Salas et al , J Virology. 67, pp 3748-55 (1993) and Mountford et al . Proc Natl. Acad. Sci. USA. 91. pp 4303-07 (1994) Use of these modified sequences is also contemplated in this invention
  • Chromaffin cells are known to secrete a number of potentially therapeutic molecules
  • a patient may be implanted with a capsule device containing a first cell type (e g , hCNTF-secreting BHK cells) If after time, the patient develops an immune response to that cell type, the capsule can be retrieved, or explanted, and a second capsule can be implanted containing a second cell type (e g , C 2 C 12 cells) In this manner, continuous provision ofthe therapeutic molecule is possible, even if the patient develops an immune response to one of the encapsulated cell types
  • a first cell type e g , hCNTF-secreting BHK cells
  • any ofthe foregoing cells may be used to deliver CNTF (and any other co-delivered molecules, as set forth above)
  • Encapsulation hinders elements ofthe immune system from entering the capsule, thereby protecting the encapsulated cells from immune destruction
  • This technology increases the diversity of cell types that can be employed in therapy
  • the semipermeable nature ofthe capsule membrane also permits the molecule of interest to easily diffuse from the capsule into the surrounding host tissue This technique prevents the inherent risk of tumor formation and allows the use of unmatched human or even animal tissue, without immunosuppression ofthe recipient
  • the implant may be retrieved if necessary or desired It is both undesirable and expensive to maintain a patient in an immunosuppressed state for a substantial period of time. Such retrievability may be essential in many clinical situations.
  • Capsules have been categorized as Type 1 (Tl), Type 2 (T2), type 1/2 (Tl/2) or Type 4 (T4) depending on their outer surface morphology
  • Tl Type 1
  • T2 Type 2
  • Tl/2 type 1/2
  • T4 Type 4
  • a biocompatible capsule means that the capsule, upon implantation in a host mammal, does not elicit a detrimental host response sufficient to result in the rejection ofthe capsule or to render it inoperable, for example through degradation.
  • an immunoisolatory capsule means that the capsule upon implantation into a mammalian host minimizes the deleterious effects ofthe host's immune system on the cells within its core
  • a variety of biocompatible immunoisolatory capsules are suitable for delivery of molecules according to this invention Such capsules will allow for the passage of metabolites, nutrients and therapeutic substances while minimizing the detrimental effects of the host immune system
  • the capsule of this invention will be similar to those described in Aebischer et al., PCT publication WO
  • Useful biocompatible polymer capsules comprise (a) a core which contains a cell or cells, either suspended in a liquid medium or immobilized within a hydrogel or extracellular matrix, and (b) a surrounding or peripheral region of permselective matrix or membrane (jacket) which does not contain isolated cells, which is biocompatible, and which is sufficient to protect isolated cells if present in the core from detrimental immunological attack
  • the core ofthe polymer capsule is constructed to provide a suitable local environment for the continued viability and function ofthe cells isolated therein
  • Many transformed cells or cell lines are most advantageously isolated within a capsule having a liquid core.
  • cells can be isolated within a capsule whose core comprises a nutrient medium, optionally containing a liquid source of additional factors to sustain cell viability and function, such as fetal bovine or equine serum
  • the core may be composed of a matrix formed by a hydrogel which stabilizes the position ofthe cells in cell clumps
  • hydrogel herein refers to a three dimensional network of cross-linked hydrophilic polymers
  • the network is in the form of a gel, substantially composed of water, preferably but not limited to gels being greater than 90% water
  • compositions which form hydrogels fall into three classes
  • the first class carries a net negative charge (e.g., alginate)
  • the second class carries a net positive charge (e.g., collagen and laminin)
  • Examples of commercially available extracellular matrix components include MatrigelTM and VitrogenTM Fibroblasts generally survive well in a positively charged matrix and are thus suitably enclosed in extracellular-matrix type hydrogels
  • the third class is net neutral in charge (e.g , highly crosslinked polyethylene oxide, or polyvinylalcohol) Any suitable matrix or spacer may be employed within the core, including precipitated chitosan, synthetic polymers and polymer blends, microcarriers and the like, depending upon the growth characteristics ofthe cells to be encapsulated
  • the capsules are immunoisolatory
  • the surrounding or peripheral region ofthe capsule should confer protection ofthe cells from the immune system ofthe host in whom the capsule is implanted, by preventing harmful substances of the host's body from entering the core ofthe vehicle, and by providing a physical barrier sufficient to prevent detrimental immunological contact between the isolated cells and the host's immune system
  • the thickness of this physical barrier can vary, but it will always be sufficiently thick to prevent direct contact between the cells and/or substances on either side ofthe barrier
  • the thickness of this region generally ranges between 5 and 200 microns; thicknesses of 10 to 100 microns are preferred, and thickness of 20 to 75 microns are particularly preferred
  • Types of immunological attack which can be prevented or minimized by the use ofthe instant vehicle include attack by macrophages, neutrophils, cellular immune responses (e g natural killer cells and antibody-dependent T cell-mediated cytolysis (ADCC), and humoral response (e g , antibody-dependent, complement-mediated cytolysis)
  • ADCC antibody-dependent T cell-mediated
  • polymers and polymer blends can be used to manufacture the capsule jacket, including polyacrylates (including acrylic copolymers), polyvinylidenes, polyvinyl chloride copolymers, polyurethanes, polystyrenes, polyamides, cellulose acetates, cellulose nitrates, polysulfones (including polyether sulfones), polyphosphazenes, polyacrylonitriles, poly(acrylonitrile/covinyl chloride), as well as derivatives, copolymers and mixtures thereof
  • the capsule can be any configuration appropriate for maintaining biological activity and providing access for delivery ofthe product or function, including for example, cylindrical, rectangular, disk-shaped, patch-shaped, ovoid, stellate, or spherical Moreover, the capsule can be coiled or wrapped into a mesh-like or nested structure If the capsule is to be retrieved after it is implanted, configurations which tend to lead to migration ofthe capsules from the site of implantation, such as spherical capsules small enough to travel in the recipient's blood vessels, are not preferred Certain shapes, such as rectangles, patches, disks, cylinders, and flat sheets offer greater structural integrity and are preferable where retrieval is desired
  • the implantable capsule is of a sufficient size and durability for complete retrieval after implantation
  • the device has a tether that aids in retrieval
  • tethers are well known in the art
  • Such macrocapsules have a core of a preferable minimum volume of about 1 to 10 ⁇ l and depending upon use are easily fabricated to have a volume in excess of 100
  • the preferred capsule will have an inner single ultrafiltration membrane with a permselective pore-size permeability range of 60-98% BSA rejection coefficient and 50-90% ovalbumin rejection coefficient
  • the fiber In a hollow fiber configuration, the fiber will have an inside diameter of less than 1500 microns, preferably less than 300-600 microns In either geometry, the hydraulic permeability will be in the range of 1 - 100 mls/min/M 2 /mmHg, preferably in the range of 25 to 70 mls/min/M 2 /mmHg
  • the glucose mass transfer coefficient ofthe capsule defined, measured and calculated as described by Dionne et al , ASAIO Abstracts, p 99 (1993), and Colton et al , The Kidney, eds , Brenner BM and Rector FC, pp 2425-89 (1981) will be greater than 10 "6 cm sec, preferably greater than 10 "4 cm sec Tl membranes may be formed by coextrusion of a polymer solution and coagulant solution through air before entering a quench bath T2 membranes may be formed by coextruding the polymer, and coagulation solutions into humidified air or a mist and then into a bath T
  • Tl/2 membranes may be formed using similar methods used to form T2 membranes
  • the mist or humidity at the coextrusion port may be controlled according to known methods to produce the desired outer surface mo ⁇ hology
  • the nozzle distance from a quench bath may be varied, according to routine methods
  • the absolute and/or relative flow rates of polymer and coagulant may be adjusted to achieve the desired outer wall surface mo ⁇ hology
  • the polymer and coagulant solution compositions and temperatures can be varied to achieve the desired outer surface wall mo ⁇ hology See WO 95/0542
  • any suitable method of sealing the capsules may be used, including the employment of polymer adhesives and/or crimping, knotting and heat sealing These sealing techniques are known in the art.
  • any suitable "dry” sealing method can also be used in such methods, a substantially non-porous fitting is provided through which the cell-containing solution is introduced Subsequent to filling, the capsule is sealed Such a method is described in copending United States application Serial No 08/082,407, herein incorporated by reference
  • the capsule is formed from a polyether sulfone hollow fiber, such as those described in United States Patent Nos 4,976,859 and 4,968,733, herein incorporated by reference
  • microcapsules for cellular delivery See, e g , United States patents 4,352,883, 4,353,888, and 5,084,350, herein incorporated by reference
  • polymer rods e g , EVA rods
  • implantation sites include the central nervous system, including the brain, spinal cord, and aqueous and vitreous humors ofthe eye
  • Preferred sites in the brain include the striatum, the cerebral cortex, subthalamic nuclei and nucleus Basalis of Maynert
  • Other preferred sites are the cerebrospinal fluid, most preferably the subarachnoid space and the lateral ventricles
  • the actual dosage can be varied by any suitable method known in the art, including, e g , by implanting a fewer or greater number of capsules We prefer between one and ten capsules
  • the dosage should be sufficient to produce a measurable level of CNTF in the CSF
  • the cell loading density may also be varied over a wide range If macrocapsules are used, preferably between 10 3 and 10 s cells are encapsulated, most preferably 10 5 to 10 7 cells are encapsulated
  • CNTF (or other suitable molecule) is delivered at a dosage sufficient to maintain a measurable concentration up to 1000 ng/ml in the CSF, preferably between 0 01 ng/ml - 1000 ng/ml in the CSF, most preferably 0 1-100 ng/ml in the CSF
  • the non- viral based vector is stably integrated into the cells, thus avoiding all independent replication events and possible transmission of the vector, 2) the semipermeable membrane surrounding the transplanted cells prevents any genetic exchange between the transfected cells and the human host cells,
  • the cells are rejected by the host immune system, 4) the expression vector constitutively produces thymidine kinase
  • HSV-tk HSV-tk
  • a linker generating a Smal site was introduced at +6 ofthe mouse MT-I promoter that extended 5' to the natural Kpnl site at about -600 This Smal site was fused to a Klenow-filled Xbal site at the 5' end of a 150 bp Xbal-EcoRI fragment ofthe murine immunoglobulin region containing parts of exons 1 and 2 encoding the signal peptide and the small intervening intron A
  • the second exon has an EcoRI site at amino acid 18 of the signal peptide
  • the human CNTF gene was obtained by PCR amplification of human DNA A 2700 bp fragment was amplified with primers that included an EcoRI site at the position ofthe natural hCNTF initiation codon and a Bgl ll site 7 bp 3' of its termination codon
  • the coding region ofthe cloned amplification product was sequenced to determine that the open reading frame was intact
  • the MT/Ig fragment was fused to the 5' end
  • the hCNTF gene was fused to the MT/Ig sequence via the EcoRI site located in the second exon at amino acid 18 of the Ig signal peptide, such that the sequence starting with amino acid 18 is Asn-Ser-Ala-Phe-Thr-Glu-His-Ser (SEQ. ID NO 2) where the underlined amino acids represent the amino terminal sequence of hCNTF after the initiating methionine is removed.
  • SEQ. ID NO 2 Asn-Ser-Ala-Phe-Thr-Glu-His-Ser
  • hGH human Growth Hormone gene
  • RP3224E2 The final pNUT-hCNTF plasmid construction, named RP3224E2, was amplified in a standard E.coli strain (HB1O1) and purified by the Qiagen-Plasmid Kit (Kontron) RP3224E2 was transfected into a line of baby hamster kidney cells
  • BHK using standard calcium phosphate methodology
  • Gene amplification was performed in increasing concentrations of methotrexate (1-200 ⁇ M) over 8 weeks to produce stable amplified cell lines
  • methotrexate (1-200 ⁇ M)
  • the engineered BHK cells were maintained in vitro in 50-200 ⁇ M MTX No loss of CNTF expression has been observed in the absence of drug selection over three months inclusive, when assayed by Northern Blot analysis, CNTF bioassay, or ELISA for CNTF portein (R&D Systems Inc )
  • the encapsulation technique allows the selection of cells over several months for stable expression ofthe DHFR gene before transplantation
  • the selected cells can then be analyzed for transgene expression by Northern and Southern blot
  • the Southern blot analysis revealed the insertion of 38 gene copies for the BHK-hCNTF-tk clone#72, the clone chosen for clinical application
  • the level of human CNTF expression from each capsule is tested by ELISA before implantation
  • the production of human CNTF was approximately 0 5 ⁇ g/24h/10 5 cells
  • the protein is biologically active as determined by a bioassay on ciliary ganglion neurons and embryonic motoneurons in vitro
  • the encapsulation procedure was as follows
  • the hollow fibers were fabricated from a polyether sulfone (PES) with an outside diameter of 720 ⁇ and a wall thickness of a 100 ⁇ m (AKZO-Nobel Wuppertal, Germany) These fibers are described in United States patents 4,976,859 and 4,968,733, herein inco ⁇ orated by reference In some studies, including the human study, we used a PES#5 membrane which has a MWCO of about 280 kd In other studies we have used, or contemplate using a PES#8 membrane which has a MWCO of about 90 kd
  • the devices used comprised
  • the components ofthe device are commercially available
  • the LCM glue is available from Ablestik Laboratories (Newark, DE), Luxtrak Adhesives LCM23 and LCM24)
  • the tether material is available from Specialty Silicone Fabricators (Robles, CA)
  • the tether dimensions are 0 79 mm OD x 0 43 mm ID x length 202 mm
  • the mo ⁇ hology ofthe device is as follows
  • the inner surface has a permselective skin
  • the wall has an open cell foam structure
  • the outer surface has an open structure, with pores up to 1 5 ⁇ m occupying 30 + 5% ofthe outer surface
  • Fiber material was first cut into 5 cm long segments and the distal extremity of each segment were sealed with a photopolymerized acrylic glue (LCM-25, ICI) Following sterilization with ethylene oxide and outgassing, the fiber segments are loaded with a suspension of 2 x 10 5 transfected cells in a collagen solution (Zyderm® soluble bovine collagen) via a Hamilton syringe and a 25 gauge needle through an attached injection port The proximal end ofthe capsule was sealed with the same acrylic glue In some studies the collagen matrix was ZyplastTM The volume ofthe device used in the human studies was approximately 15-18 ⁇ l
  • a silicone tether (Specialty Silicone Fabrication, Taunton, MA) (ID 690 ⁇ m, OD 1 25 mm) was placed over the proximal end ofthe fiber allowing easy manipulation and retrieval ofthe device
  • mice were examined for grasp activity ofthe hind paws As soon as the disease was detected (between 16 and 20 days), the mice received subcutaneous BHK-hCNTF or control BHK cell containing capsules releasing around 0 1 g/day of CNTF
  • Mice transplanted with hCNTF releasing- capsules also showed diminished motor impairment as compared to mice receiving capsules containing the parent BHK cell line
  • the BHK cells were screened for the presence of various pathogens according to the Points to Consider (PTC) cell line safety evaluated guidelines The cells were found to be free of mycoplasma, pneumonia virus, sendai virus, reovirus type 3, hantaan virus, lymphocytic choriomeningitis and simian virus type 5
  • Ganciclovir sensitivity was determined on BHK-hCNTF-tk clone#72 in comparison to the polyclonal line not containing the TK gene
  • the cells were plated at approximately 75,000 cells/well and treated with 3 concentrations (0 02, 0.2 and 2 ⁇ M) of ganciclovir
  • 3 concentrations (0 02, 0.2 and 2 ⁇ M) of ganciclovir
  • cells were treated with fresh ganciclovir and the effect was determined after 4 days
  • No effect of ganciclovir was observed on the polyclonal line of BHK-hCNTF cells (tk-) at any concentration
  • the in vivo efficacy ofthe tk system was tested in a nude mouse model
  • the nude mouse is a highly stringent model allowing to test the sensitivity ofthe BHK-hCNTF-tk clone#72 cells in the absence of T-cell mediated immunological response Thirty animals were used, 10 for each of the following conditions
  • Group 1 received ganciclovir 50 ⁇ g/kg, by injection two times a day (BID) for 5 days
  • Group 2 received inoculations of 1 x IO 6 BHK-hCNTF-tk cells suspended in 100 ⁇ l and saline (vehicle) injections BID for 5 days
  • Group 3 received inoculations of BHK-hCNTF-tk cells (1 x 10 6 /100 ⁇ l) and ganciclovir injections (50 mg/kg) BID for 5 days
  • the animals were weighed once per week during the entire 4 week evaluation The animals were also monitored for the appearance ofthe tumor nodules as well as for their overall health Histological analysis for the presence of subcutaneous nodules on their bodies were performed immediately after sacrifice.
  • the rats weighed around 300 gr they received more than 400 times more CNTF than the amount we proposed delivering to humans
  • the capsules were placed in the subarachnoid space over the spinal cord through a laminectomy performed at the LI-L2 level The animals were then closely observed during the course ofthe implantation monitoring rectal temperature, weight and general behavior Cerebrospinal fluid (CSF) was collected at the time of sacrifice for CNTF determination through an occipital tap
  • CSF Cerebrospinal fluid
  • the retrieved capsules were fixed in a 4 % paraformaldehyde (PAF) solution
  • the animals were transcardially perfused with a 4 % PAF solution
  • the spinal cord was inspected in situ and dissected for complete histologic analysis Biocompatibility and viability ofthe encapsulated cells was assessed on glycolmethacrylate sections
  • Potential toxicity of hCNTF on nervous tissue was assessed on frozen sections of the spinal cord analyzing the general mo ⁇ hology by Nissl stain, the astrocytic reaction by GFAP and the microglia reactivity by immunohistochemistry The following results have been obtained
  • the CNTF released by each capsule was measured one day prior to implantation by ELISA Five rats were implanted with these capsules for 7 days No adverse effect was observed on the general behavior ofthe implanted animals and no significant variation ofthe animal's weight or temperature was seen
  • the intraventricular delivery of 2 4 to 60 ⁇ g of BDNF per day induces a 10 to 20 % weight loss over one week (Yan, Q , et al , "The biological responses of axotomized adult motoneurons to brain-derived neurotrophic factor," J Neurosci. 14, pp 5281- 5291, (1994))
  • More CNTF was released at explant as compared to preimplant value (0.37 + 0 1 1 vs 0.62 + 0.15 ⁇ g/day respectively)
  • BHK/CNTF cells The 5 cm long capsules were implanted in the sheep lumbar subarachnoid space as previously described (Joseph, J M , et al , "Transplantation of encapsulated bovine chromaffin cells in the sheep subarachnoid space a preclinical study for the treatment of cancer pain," Cell Transpl. 3, pp 355-364 (1994))
  • the cell density was 50 x 10 3 cells/ ⁇ l for all animals
  • the capsules were left in place for one month in all animals CSF was collected at the time of implant, 8 days postimplantation and at the explantation time Both cellularity and protein level were analyzed
  • the histological analysis ofthe explanted capsules showed the presence of viable BHK cells surrounding a core of non-viable cells No systemic toxicity as assessed by behavior, rectal temperature or weight measurement was observed A strong GFAP reactivity was present in animals which had adhe ⁇ ng or intramedullary implants In contrast, the GFAP reactivity was small in animals with "free floating" capsules Nissl stain showed normal neuronal cellularity in all animals
  • the CSF analysis revealed both an increase of cellularity and protein concentration 8 days post-implantation as compared to the pre-implant level This increase however returns to normal at 4 weeks Human Clinical Trial
  • the device was to be explanted and measured for cell viability and CNTF production
  • a new device could be implanted and the patient could then be followed for a further 6 months
  • Plasma as well as CSF levels of CNTF were measured and serum samples were also drawn and analyzed to determine whether an immune reaction was generated against the BHK cells that were in the capsule and producing CNTF Patients were rechecked for performance against the motor scale that had been used to evaluate them during the three month run in period and queried for side effects
  • a cranio-caudal skin incision of 3 cm is performed at the L4-L5 level Section the subcutaneous tissue down to the dorsal fascia Puncture the subarachnoid space with a 25G Tuohy needle and withdraw 12 ml of CSF.
  • the skin is re-opened at the same place and the 4-0 polypropylene section
  • the capsule is retrieved by gentle pulling on the silicone tether
  • the biocompatibility, the histology and the CNTF release ofthe explanted capsule are examined before re-implanting of a new device in the patient, following the same protocols
  • TQNE Tufts Quantitative Neuromuscular Exam
  • the battery includes testing of five major functional areas bulbar, respiratory, arm, leg, and fine motor activities
  • the TQNE test battery consists of 28 qualitative items that assess motoneuron function at different levels of the neuraxis In this way, one can predict the patient's individual course ofthe disease on the basis of 3 successive tests 1 month apart
  • the capsules Upon explant, the capsules had viable cells still producing CNTF
  • Fig 7 shows the Norris scores on patient 1 (Panel A), patient 2 (Panel B), patient 3 (Panel C), patient 4 (Panel D), and patient 5 (Panel E)
  • the expression vector RP3224E2 contains the entire human CNTF gene as described above
  • the RP3224D plasmid which is identical to RP3224E2, but carries no HSV-tk gene, was modified as follows RP3224D was digested with the restriction enzymes Sail and Clal to remove the dihydrofolate reductase gene (DHFR) as well as the Hepatitis virus B 3 ' untranslated region (HBV-3 ' UTR)
  • the purified vector was filled in by using the Klenow fragment
  • the neomycin resistance gene and the SV40 promoter were excised from a pNUT/neo plasmid (Dr E Baetge, CytoTherapeutics, Inc., Buffalo, USA) with the restriction enzymes Hindlll and BamHI
  • the isolated fragment was blunted by filling in the protruding ends with T4 DNA polymerase and subcloned into the Sall/Clal RP3224D vector
  • C 2 C 12 mouse myoblasts derived from leg skeletal muscles of an adult C3H mouse were obtained from American Type Culture Collection (ATCC, CRL 1772, Rockville, MD)
  • C 2 C 12 cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS), 2 mM L-glutamine, 4 5 g/1 glucose, 1 OOU/ml penicillin, 1 OOU/ml streptomycin
  • DMEM Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • FCS fetal calf serum
  • Differentiation of C 2 C ⁇ myoblasts into post-mitotic myotubes was carried out by exposing the confluent myoblast cultures to DMEM with 2% fetal calf serum for 3-4 days The cells were maintained in the incubator at 37°C and 5% CO 2
  • the plasmid pNUT-hCNTF-NT was transfected into the
  • a BamHI fragment (546 bp) from the RP3224E2 plasmid corresponding to the 3' end ofthe hCNTF gene was subcloned into pBluescriptSK " plasmid (Stratagene)
  • the plasmid pSp65BAC was obtained by inserting a 200 bp (Pstl-Bglll) fragment ofthe mouse ⁇ -actin cDNA into the pSp65 vector (Promega Biotech) (Jongeneel et ai, J Immunol .
  • RNA probes were linearized using appropriate enzymes and transcribed in vitro with T3 or Sp6 RNA polymerase (Promega Biotech) and a- 32 P-UTP (Amersham) Fifteen ⁇ g of total cytoplasmic RNA were used for Northern blot analysis as described previously (Deglon et al., Eur. J. Biochem..
  • the filter was hybridized with 1 x 10 6 counts per minute per ml (cpm/ml) of anti-sense RNA probes for 48 hours at 55°C in the hybridizing solution
  • the filters were washed 4 times for 15 minutes each time in 0 1 x SSC, 0 1% SDS at 60°C
  • Densitometric analysis of several autoradiograms with different exposure times was performed using a ScanJet Ilex scanner (Hewlett Packard) and the DeskScan II and Scan Analysis softwares Normalization ofthe steady state level of CNTF and actin expression was performed using the 28 S RNA probe
  • hCNTF released from the various C 2 C 12 clones was determined in vitro using cultures of ventral spinal cord from El 4 embryonic rats Motoneurons were prepared as described by Martinou et al, Neuron. 8, pp 737-44 (1992)
  • a dose response curve with recombinant hCNTF (Calbiotech, Mountain View, CA) was used as a positive control
  • Conditioned medium was obtained by exposing either the parent C 2 C ⁇ cells or the hCNTF-C 2 C 12 cells (1 8xl0 5 cells per well) to 2 ml of embryonic motoneuron culture medium for 12 hours
  • Embryonic rat motoneuron cultures were grown in the presence of 50 ⁇ l of conditioned medium which was replaced every 2 days
  • the choline acetyltransferase (ChAT) present in the cultures was determined by measuring the production of 3 [H] acetylcholine from 3 [H] acetylcoenzyme A (Beretta and Zurn, De
  • hCNTF-C 2 C 12 Parent C 2 C I2 cells or hCNTF-C 2 C 12 (clone #15) cells were plated at a density of 1 x 10 5 cells per well for twelve hours on 6-weIl tissue culture dishes (Costar) in PC-1 medium, a serum free medium containing human recombinant proteins (Hycor Biomedical Inc., CA). Conditioned media were obtained by incubating the cells encapsulated or not in 2 ml of fresh PC-1 medium for 30 minutes. These samples were then stored at -20°C until tested using an ELISA kit for hCNTF (R&D Systems, Minneapolis, MN)
  • Sensitivity to ganciclovir Control C 2 C 12 or C 2 C 12 cells transfected with the pNUT-hCNTF-DNT vector were plated at a density of 2 x 10 5 cells per well on 6- well tissue culture dishes (Costar) C 2 C 12 myoblast differentiation was induced by exposing the culture to DMEM containing 2% FCS for 3 days Non-differentiated C 2 C 12 and hCNTF-C 2 C 12 were plated at a density of 1 x IO 4 cells per well at the beginning of ganciclovir treatment Fresh ganciclovir solution (Cytovene* Svntex) at a final concentration of 5 ⁇ M was added to the cultures for 5 days Results were evaluated by microscopic examination E ⁇ capsulation
  • Control and transfected C 2 C 12 cells were harvested using 0 125% trypsin Dilutions were made to achieve a suspension of 1 xlO 5 cells/ ⁇ l in 1 75 mg/ml Zyderm (Collagene S A , Lausanne, Switzerland) The cell suspension was then injected into microporous polyethersulfone hollow fibers (OD 550 ⁇ m, ID 350 ⁇ m) (Akzo Nobel Faser AG, Wupperthal, Germany) One centimeter long capsules were made by cutting them to appropriate lengths and heat sealing the ends Each capsule contained approximately 2xl0 5 cells at the time of encapsulation Capsules were kept in DMEM medium containing 10% FCS in an incubator at 37°C and 5% CO 2 for two days prior to differentiation in low serum medium for 3 days Capsules were then transfered to PC- 1 medium and subsequently tested using the in vitro bioassay and the ELISA assay
  • the bioactivity of the produced hCNTF was evaluated using embryonic rat ventral spinal cord cultures, and measuring changes in neuronal ChAT activity in the presence or the absence ofthe ciliary neurotrophic factor. Results confirm that the transfected C 2 C 12 myoblasts produce a bioactive hCNTF. ChAT activity in cultures exposed to conditioned media from hCNTF-C 2 C 12 cells was comparable to that obtained following a single application of 20 ng/ml recombinant hCNTF. The ChAT bioassay also demonstrates that C 2 C ]2 cells continue to secrete hCNTF following differentiation
  • HSV-tk he ⁇ es simplex virus thymidine kinase gene
  • the neonatal rat axotomy model was used to evaluate the efficacy of our hCNTF delivery system Immediatly following the axotomy ofthe facial nerve, newborn rats (P2) were implanted subcutaneously with polymer capsules containing either control C 2 C, 2 cells or hCNTF-transfected myoblasts
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:l:
  • GGTACCTTGG TTTTTAAAAC CAGCCTGGAG TAGAGCAGAT GGGTTAAGGT GAGTGACCCC 60
  • TCGTAAACTC CAGAGCAGCG ATAGGCCGTA ATATCGGGGA AAGCACTATA GGGACATGAT 360
  • GACAGTGGCA ATCACTTTGC CTTTCTTTCT ACAGGGGTGA ATTCGGCTTT CACAGAGCAT 780 TCACCGCTGA CCCCTCACCG TCGGGACCTC TGTAGCCGCT CTATCTGGCT AGCAAGGAAG 840
  • ATACTCTTCC TTTTTCAATA TTATTGAAGC ATTTATCAGG GTTATTGTCT CATGAGCGGA 7080
  • TTTACTTGCC AATTCCGGTT GTTCAATAAG TCTTAAGGCA
  • TCATCCAAAC TTTTGGCAAG 8880 AAAATGAGCT CCTCGTGGTG GTTCTTTGAG TTCTCTACTG AGAACTATAT TAATTCTGTC 8940

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Abstract

Un procédé de traitement de la sclérose latérale amyotrophique chez un patient souffrant de cette maladie consiste à administrer directement dans le système nerveux central dudit patient, une dose suffisante de facteur neurotrophique ciliaire (CNTF) pour maintenir dans le liquide céphalo-rachidien un niveau mesurable de CNTF pouvant atteindre jusqu'à 1000 ng/ml de préférence 0,1-100 ng/ml, ou une dose de hCNTF comprise entre 1-10000 ng/jour, de préférence comprise entre 250-1000 ng/jour.
PCT/US1996/015824 1995-10-02 1996-10-02 Procede de traitement de la sclerose laterale amyotrophique WO1997012635A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU72049/96A AU7204996A (en) 1995-10-02 1996-10-02 Method for treating amyotrophic lateral sclerosis

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US53733895A 1995-10-02 1995-10-02
US08/537,338 1995-10-02

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Publication Number Publication Date
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WO (1) WO1997012635A1 (fr)

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WO1999043813A1 (fr) * 1998-02-27 1999-09-02 Regeneron Pharmaceuticals, Inc. Facteur neurotrophique ciliaire modifie, procede de fabrication et procedes d'utilisation
WO2005060992A1 (fr) 2003-12-24 2005-07-07 The Walter And Eliza Hall Institute Of Medical Research Agents therapeutiques et utilisations
WO2005108981A1 (fr) 2004-05-12 2005-11-17 The Walter And Eliza Hall Institute Of Medical Research Procede d'isolement de cellules
US7125837B1 (en) 1999-02-26 2006-10-24 University Of Utah Research Foundation Elastin-based compositions
WO2008011006A2 (fr) 2006-07-18 2008-01-24 University Of Utah Research Foundation Procédé pour traiter la douleur et rechercher systématiquement des composés analgésiques
EP2119728A1 (fr) 2002-11-29 2009-11-18 The Corporation Of The Trustees Of The Order Of The Sisters Of Mercy In Queensland Agents thérapeutiques et de diagnostic
EP2407484A2 (fr) 2005-06-24 2012-01-18 The Walter And Eliza Hall Institute Of Medical Research Molécules thérapeutiques de pro-apoptose de type BH3 et procédés pour les générer et/ou les sélectionner
WO2014194284A1 (fr) 2013-05-31 2014-12-04 Mcintosh J Michael Peptides de conotoxine, compositions pharmaceutiques et leurs utilisations
WO2017070738A1 (fr) 2015-10-27 2017-05-04 The University Of Queensland Procédé de traitement et agents utiles audit procédé

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999043813A1 (fr) * 1998-02-27 1999-09-02 Regeneron Pharmaceuticals, Inc. Facteur neurotrophique ciliaire modifie, procede de fabrication et procedes d'utilisation
US7125837B1 (en) 1999-02-26 2006-10-24 University Of Utah Research Foundation Elastin-based compositions
EP2119728A1 (fr) 2002-11-29 2009-11-18 The Corporation Of The Trustees Of The Order Of The Sisters Of Mercy In Queensland Agents thérapeutiques et de diagnostic
WO2005060992A1 (fr) 2003-12-24 2005-07-07 The Walter And Eliza Hall Institute Of Medical Research Agents therapeutiques et utilisations
WO2005108981A1 (fr) 2004-05-12 2005-11-17 The Walter And Eliza Hall Institute Of Medical Research Procede d'isolement de cellules
EP2407484A2 (fr) 2005-06-24 2012-01-18 The Walter And Eliza Hall Institute Of Medical Research Molécules thérapeutiques de pro-apoptose de type BH3 et procédés pour les générer et/ou les sélectionner
WO2008011006A2 (fr) 2006-07-18 2008-01-24 University Of Utah Research Foundation Procédé pour traiter la douleur et rechercher systématiquement des composés analgésiques
WO2014194284A1 (fr) 2013-05-31 2014-12-04 Mcintosh J Michael Peptides de conotoxine, compositions pharmaceutiques et leurs utilisations
WO2017070738A1 (fr) 2015-10-27 2017-05-04 The University Of Queensland Procédé de traitement et agents utiles audit procédé

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