Patent Application
20230257436
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 15, 2020, is named P9590US00_ST25.txt and is 144,484 bytes in size.
The present invention concerns methods and compositions for novel biomarkers and treatments including, but not limited to, recombinant proteins and gene- and cell-based therapies, in particular, combinatorial therapies for delivery of progranulin, prosaposin, a complex of progranulin and prosaposin (also referred to herein as “progranulin/prosaposin complex(es)”), alpha-synuclein targeting antibodies and their neurorestorative factors, including, but not limited to, the neurorestorative factor GDNF, in different combinations, for the treatment of neurodegenerative diseases and lysosomal storage disorders. In another aspect, the invention relates to cell lines expressing alpha-synuclein targeting antibodies, GDNF, progranulin, prosaposin, a complex of progranulin and prosaposin, methods of manufacture of such, methods of monitoring such in, e.g., human serum and CSF, and the use of both recombinant factors as therapeutics or a cell line inside of an implantable cell device for the delivery of the alpha-synuclein targeting antibodies, GDNF, progranulin, prosaposin and progranulin/prosaposin complex to a patient.
Frontotemporal dementia (FTD) is a neurological disorder characterized by the atrophy of the frontal lobe and/or anterior temporal lobe as visualized by structural magnetic resonance imaging or positron emission tomography. FTD represents an estimated 10%-20% of all dementia cases. It is recognized as one of the most common presenile dementias, affecting between 15-22 per 100,000 people. Signs and symptoms typically manifest in late adulthood, commonly between the ages of 45 and 65. Signs and symptoms typically include one or more changes in social behavior and conduct, loss of social awareness and poor impulse control, impaired verbal comprehension, progressive, non-fluent aphasia, and marked changes in behavior. As the disease progresses, patients may present symptoms comparable to Alzheimer's Disease, such as loss of executive functioning and working memory. Currently, there is no cure for FTD aside from treatments to manage behavioral symptoms, typical selective serotonin reuptake inhibitors.
One mechanism of FTD is mutations in the granulin (GRN) gene. Haploinsufficiency in GRN readily monitored as decreased extracellular levels of progranulin (PGRN), the precursor form of granulin, typically results in an inheritable form of FTD, and complete loss of PGRN, also leads to a lysosomal storage disorder, neuronal ceroid lipofuscinosis (NCL). Extracellular PGRN is taken up by neurons and transported to the lysosomes via different mechanisms. PGRN also facilitates neuronal uptake and lysosomal delivery of prosaposin (PSAP), the precursor of saposin peptides that are essential for lysosomal glycosphingolipid degradation. Additionally, PGRN mutant neurons have reduced lysosomal GCase activity, lipid accumulation and increased insoluble alpha-synuclein. Brain tissue samples from patients with FTD show reduced levels of PSAP in neurons. Decreased cellular uptake of extracellular PGRN and reduced PGRN-mediated PSAP lysosomal trafficking may therefore be an underlying disease mechanism for NCL and FTD due to GRN mutations. To this end, no one has monitored or characterized the PGRN/PSAP complex in plasma or CSF, and to what extent its expression levels may be altered in disease. There is a need in the art for specific assays to determine the absolute and relative levels of PGRN, PSAP and/or PGRN/PSAP as individual fluid biomarkers and pathway biomarker profiles, respectively, for diagnosis, prognosis, therapy development and to monitor treatment responses.
In addition to regulating each other's expression levels, PGRN and PSAP interact physically to facilitate each other's lysosomal trafficking and PGRN-PSAP interaction is important for maintaining proper lysosomal function in the brain. To date, however, no one has shown that supplementation by either extracellular PGRN or PGRN-PSAP complexes can prevent or treat NCL and FTD. There is a need in the art for an effective method to produce PGRN-PSAP complexes in a way that also allows transport of these molecular entities to the brain where they can protect against neurodegeneration.
In a first aspect, the invention relates to cell lines which express one or several alpha-synuclein targeting antibodies or antibody fragments, and/or progranulin, and/or prosaposin, and/or GDNF and their subpeptides and derivatives. In a preferred embodiment, the cell line is genetically modified to produce these factors simultaneously, for example, by the insertion of plasmids into the cell line. In various embodiments, the alpha-synuclein targeting antibodies/fragments, GDNF and progranulin and prosaposin are expressed as polypeptides, subpeptides, RNA, or exosomal RNA.
Many different cell types may be encapsulated in the devices according to the present invention. These include well-known, publicly available immortalized cell lines, spontaneously immortalized cell lines as well as dividing primary cell cultures. As cell lines in some embodiments are to be transfected or transduced, clones have to be selected, expanded and cell banked, it is preferable that the cells or cell lines are capable of undergoing a significant number of divisions.
Cell lines with long term propagation potential may be created from a wide variety of cells, including progenitor and/or precursor cells. Also suitable are stem cells including pluripotent and multipotent stem cells, embryonal stem cells, neural stem cells, and hematopoietic stem cells.
Cell lines of the invention include Mouse myeloma cells (NS0), Chinese hamster ovary cells (CHO); CHO-K1; baby hamster kidney cells (BHK); mouse fibroblast-3T3 cells; African green monkey cell lines (including COS-1, COS-7, BSC-1, BSC-40, BMT-10 and Vero); mesenchymal chondroSarcoma-1 (MCS); rat adrenal pheochromocytoma (PC12 and PC12A); AT3, rat glial tumor (C6); rat neuronal cell line RN33b; rat hippocampal cell line HiB5; growth factor expanded stem cells; epidermal growth factor (EGF)-responsive neurospheres; basic fibroblast growth factor-responsive (bFGF-responsive) neural progenitor stem cells derived from the CNS of mammals; foetal cells; primary fibroblasts; Schwann cells; astrocytes; β-TC (ATCC CRL-11506) cells; human liver cancer cell line Hep-G2 striatal cells; oligodendrocytes and their precursors; mouse myoblast cells-C2C12; human glial-derived cells-Hs683; human glial-derived cells-A172; HEI193T cell line; porcine glioblasts; neuronal cells; neurons; astrocytes; interneurons; chondroblasts isolated from human long bone; human embryonic kidney cells 293 (HEK293); human cell line HeLa; rabbit corneal-derived cells (Statens Seruminstitut Rabbit Cornea (SIRC)); Human corneal derived cells, human choroid plexus cells, human induced pluripotent stem cells (iPS) cell derived cell lines, human neurotrophin 3 (NT3) cells, ARPE-19, CAC cells, immortalized human fibroblasts (MDX cells), telomerase immortalized human RPE cell lines such as hTERT RPE-1, mesenchymal stem cells (MSC).
Preferred cell lines for mammalian recombinant production include ARPE-19, CHO, CHO-1, HEI193T, HEK293, COS, NS0, C2C12, and BHK cells.
In a preferred embodiment, the cell line comprises up to four expression constructs; a first expression construct which expresses progranulin polypeptide, a progranulin gene, progranulin RNA or exosomal RNA encoding progranulin and a second expression construct which expresses prosaposin polypeptide, a prosaposin gene, prosaposin RNA or exosomal RNA encoding prosaposin and a third expression construct which expresses a gene, RNA or exosomal RNA encoding an alpha-synuclein antibody or antibody fragment, and a fourth expression construct which expresses GDNF RNA or exosomal RNA encoding GDNF. In an embodiment of the instant invention, the expression constructs comprise plasmids. In a further embodiment, the plasmids may comprise a transposon system such as Sleeping beauty transposase.
Progranulin or Prosaposin produced by cell lines of the invention may further comprise the fragment crystallizable region (Fc region) of an antibody for the purpose of enhancing the distribution and uptake of progranulin, prosaposin and a progranulin/prosaposin complex in the central nervous system. In various embodiments, the prosaposin-Fc or progranulin-Fc region combination comprises a fusion protein, fusion gene or a fusion RNA.
Progranulin and prosaposin expressed by cell lines of the invention typically form a complex, either before or after secretion from the cell line. This complex may be a heterodimer of progranulin and prosaposin.
In an embodiment of the instant invention, the cell lines of the invention further express a factor which stimulates secretion of progranulin or prosaposin from the cell line.
In a preferred embodiment, cell lines of the instant invention are contained within an implantable cell device that is then inserted into a patient in need of treatment. Examples of such a cell device can be found, generally, in U.S. Pat. Nos. 8,741,340; 9,121,037; 9,364,427; 9,669,154; 9,884,023; 10,835,664 and 10,888,526, all of which are hereby incorporated by reference. Such a device, when implanted inside of a patient, allows the alpha-synuclein antibody or antibody fragment, progranulin and prosaposin and GDNF secreted by the cell line to be efficiently delivered to the patient without the need for repeated traumas. As the factors are continuously produced, there is no need for formulation buffers and protein stability concerns. The stable cell line is also considered a single drug substance while secreting more than one effector molecule, which allows for new therapeutic interventions in difficult to treat diseases.
In a preferred embodiment, the implantable cell device comprises a semi-permeable membrane permitting the diffusion of molecules secreted from the cell line situated within said implantable cell device through said membrane. In a further embodiment, the semi-permeable membrane is immune-isolating to protect the cell line within from the patient's immune system. In another preferred embodiment, the implantable cell device comprises a matrix disposed within the semi-permeable membrane to promote efficient growth and survival of the cell line enclosed within.
In an embodiment, the implantable cell device may further comprise a means to implant the device inside of a patient in need of treatment. This implanting means may be a catheter. The device may be implanted into the patient in various tissue compartments and preferably intrathecally, intracerebroventricularly, or intracerebrally. Preferred targets for implantation include the striatum, the spinal canal and subarachnoid space of the patient.
In an embodiment, the implantable cell device may further comprise a vehicle to facilitate delivery of alpha-synuclein antibodies or antibody fragments, progranulin and prosaposin from the cell line to the desired location within the patient's body. In various embodiments, the vehicle is a pump or a syringe or associated catheter systems.
Cell lines of the invention may be useful in the treatment of neurological diseases or disorders, in particular, lysosomal storage disorder or neurodegenerative diseases that are disorders characterized by multiple pathologies. Neurological disorders treatable by cell lines of the invention include, but are not limited to, frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), limbic-predominant age-related TAR DNA-binding protein-43 (TDP-43) encephalopathy (LATE), Lewy body dementia (LBD), Parkinson's disease (PD), Multiple system atrophy (MSA) and lysosomal storage disorders. The lysosomal storage disorders that may be treated using the cell lines of the invention include, but are not limited to, Gaucher's disease, atypical Gaucher's disease, metachromatic leukodystrophy, Krabbe disease, Kyoto encyclopedia of genes and genomes (KEGG) disease, neuronal ceroid lipofuscinosis (NCL), Mucopolysaccharidosis III and IV, Tay-Sachs disease, Farber's disease, and combinations thereof.
Alpha-synuclein antibodies or antibody fragments and progranulin, prosaposin and complexes of progranulin and prosaposin produced by cell lines of the invention may also be purified for use as a therapeutic in the treatment of a neurological disorder. In this embodiment, the cell lines of the invention may be contained inside of a bioreactor to produce large quantities of alpha-synuclein antibodies or antibody fragments, progranulin, prosaposin and progranulin/prosaposin complexes. In a further embodiment, the progranulin and progranulin/prosaposin complexes are purified by several biochemical and chromatographic methods including, but is not restricted to, salt precipitation, protein A affinity chromatography, gel filtration and ion exchange chromatography. In yet a further embodiment, recombinant proteins isolated from cell lines of the invention can be administered as a therapeutic to a patient in need of treatment for a neurological disorder, as defined above. In various further embodiments, the therapeutic is administered by the use of a pump, syringe, or catheter system.
The inventors have further discovered that the complex of progranulin and prosaposin is present in body fluids, thus making it possible to monitor with inverted immune-based methods such as ELISA recognizing the complex. The absolute levels of extracellular progranulin/prosaposin complex levels may be a useful biomarker in the diagnosis and for the monitoring of drug exposure and treatment responses. In addition, the ratio of un-complexed progranulin or un-complexed prosaposin compared to progranulin/prosaposin complexes present in a fluid sample from a patient may provide important information and be a useful biomarker in the diagnosis of, but not restricted to, a neurological disorder or a means to assess the prognosis and progression of a neurological disorder, especially after treatment begins. The biomarkers of the instant application may also be used in the diagnosis and for the monitoring of an inflammatory disease, cancer and obesity-associated pathologies. Inflammatory diseases include, but are not limited to, cholelithiasis, fatty liver disease, endometriosis, inflammatory bowel disease, asthma, rheumatoid arthritis, chronic peptic ulcer, periodontitis, Crohn's disease, sinusitis, hepatitis, cardiovascular disease, arthritis, chronic obstructive pulmonary disease, encephalitis, meningitis, neuritis and pancreatitis. Obesity-associated pathologies include, but are not limited to, Type 2 diabetes mellitus, Type 1 diabetes, hyperlipidemia, insulin insensitivity, hyperglycemia, hyperinsulinemia, hypoinsulinemia, dyslipidemia, hypertension and atherosclerosis.
In various embodiments, the concentrations of un-complexed progranulin, un-complexed prosaposin, and progranulin/prosaposin complexes are determined by enzyme-linked immunosorbent assay (ELISA) or any other immune-based assay principles, such as electrochemiluminescence, e.g., the Meso Scale Discovery® technology (Meso Scale Diagnostics®, Rockville, Md.), Simoa® technology (Quanterix™ Corporation, Billerica, Mass.), HTRF® (homogenous time resolved fluorescence)(Cisbio Bioassays Societe Par Actions Simplifee a Associe Unique France Parc Marcel Boiteux B.P., Codolet, FR), Alphascreen® (PerkinElmer®, Waltham, Mass.) and/or a proximity ligation assay, however, alternative analytical methods may also be used. In various embodiments, the fluid sample may be plasma, cerebrospinal fluid, saliva, tear drops or urine.
There is a decrease in activity of the lysosomal enzyme glucocerebrosidase (GCase) encoded by the gene GBA1 in the brains of patients with frontotemporal dementia (FTD) and GRN-related frontotemporal dementia, Lewy body dementia associated with GBA1 mutations (LBD/GBA1), in Parkinson's Disease (PD) and in Gaucher's disease. Restoration of GCase activity is a major therapeutic goal for these indications. In addition, mutations in prosaposin have been linked to autosomal dominant inherited PD. Similarly, several damaging mutations in the progranulin encoding gene have been associated with PD. Extracellularly administered recombinant progranulin (PGRN), prosaposin (PSAP) and recombinant PGRN+PSAP complexes are internalized and colocalizes with GCase in lysosomes in human fibroblasts and increase GCase activity in primary cortical neurons (target cell type for FTD, LBD, advanced PD and ALS). In addition, conditioned media derived from ARPE-PGRN, ARPE-PSAP, and ARPE-PGRN+PSAP cells i.e., the therapeutic formulation of ECB-PGRN, ECB-PSAP and ECB-PGRN+PSAP therapies, increase GCase activity in primary cortical neurons. The data supports the use of either recombinant PGRN, PSAP or PGRN/PSAP or their corresponding ECB-therapies for stimulating and rescuing GCase activity in different human disease.
For the synucleinopathies LBD and PD, there is a strong link between decreased GCase activity and increased alpha-synuclein pathology/Lewy body pathology development. There is a strong rationale to combine GCase stimulation with an immunotherapy that targets alpha-synuclein misfolding in a single therapy. Importantly, the detrimental result of impaired GCase activity appears not restricted to Lewy body (LB) formation, but also impacts neuronal health in other ways leading to fatal consequences. This is suggested by the fact that disease progression, development of dementia and even lethality, besides accelerated Lewy body formation, is more aggressive and frequent in PD/GBA1 compared to PD without GBA1 mutations. Thus, multiple, therapeutic benefits, as a consequence of enhancing GCase activity combined with alpha-synuclein targeting immunotherapy, are expected. The novel alpha-synuclein targeting antibody fragments of the instant application were designed to interfere with alpha-synuclein pathology development at several levels: inhibit aggregation, bind to multiple alpha-synuclein species (monomers, oligomers, fibrils) and to exhibit a broad epitope coverage, in order to be as efficient as possible in blocking the development of alpha-synuclein pathology and to induce a sink effect. (See Methods point 7 that demonstrates the feasibility, proof, of generation of a clonal ARPE-19 cell line secreting PGRN, PSAP and an anti-alpha-synuclein targeting antibody fragment.) This cell line evidences the therapeutic effects in 8-12 week studies in two rat animal models of PD (Parkinson alpha-synuclein and 6-OHDA models). To this end, the PGRN, PSAP and PGRN+PSAP+anti-alpha-synuclein therapies have a positive impact on behavior in both models. As discussed, the GCase stimulatory factors progranulin and prosaposin and the anti-alpha-synuclein immunotherapy, mediate neuroprotection at different levels of the PD and LBD pathological cascades. In addition to these activities, there is a need to restore function of already damaged neurons. A therapeutic mediating neurorestorative activity would complement the aforementioned neuroprotective therapeutics in order to achieve an as clinically meaningful therapy as possible. GDNF is a secreted factor that has been demonstrated to mediate neurorestorative activity in animal models and in some patients. One embodiment of the claimed invention is directed to a unique therapy composed of multiple neuroprotective activities combined with neurorestorative activity, all in a single therapy. As demonstrated by the data set forth in
GRN-related frontotemporal dementia (FTD) is caused by haploinsufficiency in the secreted factor progranulin, which mediates signaling both extracellularly via different cell surface receptors and intracellularly, at the level of the lysosome where it regulates multiple factors such as Cathepsin D, PSAP and GCase. To restore PGRN signaling is a primary therapeutic goal in GRN-related FTD. PGRN deficiency in FTD/GRN is accompanied by decreased neuronal PSAP levels as well as reduced GCase activity. To what extent the levels of intracellular PGRN/PSAP complexes are affected in FTD/GRN, or to what extend the disease impacts the extracellular levels of free PSAP and the PGRN/PSAP complex are not known.
PGRN/PSAP complexes mediate neurotrophic activity:
Neuronal ceroid lipofuscinosis (NCL) is a lysosomal storage disorder caused by deficiency in PGRN (100% deficiency). Extracellularly administered PGRN or PGRN/PSAP complexes are internalized and localized to lysosomes. In addition, conditioned media from ARPE-PGRN and ARPE-PGRN+PSAP brings the same activity, thus, ECB-based administration of PGRN or PGRN/PSAP is used to rescue lysosomal PGRN signaling in NCL.
In ALS, FTD and AD, TDP pathology is the most common proteinopathy. PGRN deficiency in FTD/GRN results in TDP pathology associated neurodegeneration. Crossing a mouse model of ALS/TDP with mouse overexpressing PGRN resulted in a less severe ALS-like phenotype, i.e., PGRN appears to be therapeutic for TDP associated ALS. Based on this prior art, PGRN/PSAP maybe used as a therapeutic for ALS/TDP, as well as for FTD/TDP and AD. In particular, IT administration of PGRN or PGRN/PSAP, targeting the spinal canal and cortex, key regions in ALS pathogenesis (and FTD, LBD and AD), holds great promise as an effective treatment. The data set forth in
ARPE-19 cells (ATCC®, Manassas, Va.) were grown to 70% confluency in F12/DMEM media (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12, Thermo Fisher Scientific®, Waltham, Mass.) (Gibco®, cat no. 31331-028, supplemented with 10% FCS and penicillin/streptomycin (PEST)) (hereinafter referred to as “complete media”). On the day of transfection, the cell media was replaced with serum free media and the cells were transfected with a plasmid constitutively expressing the consensual human progranulin gene under a chicken beta-actin promoter and CMV enhancer. The recombinant expression construct also contained the neomycin selection gene and was flanked by sleeping beauty transposable elements. Transient expression of a sleeping beauty transposase was used to stably integrate copies of the PGRN cDNA transgene construct (Sleeping beauty transposon system). The plasmids were introduced using Promega® Fugene 6® transfection kit (Promega® Corporation, Madison, Wis.), according to the manufacturer's instructions. Forty-eight (48) hours post transfection, the cells were split 1:10 and seeded in 10 cm2 tissue culture dishes in the presence of complete media supplemented with 800 μg/ml Geneticin® (Thermo Fisher Scientific®, Waltham, Mass.) to select clones expressing the neomycin selection marker. Fourteen (14) days later, individual colonies were harvested and expanded in complete media supplemented with 800 μg/ml Geneticin® (Thermo Fisher Scientific®, Waltham, Mass.). The different clonal cell lines were analyzed for secreted PGRN using the R&D Systems® anti-human PGRN DuoSet® kit (R&D Systems®, Minneapolis, Minn.). Moreover, ARPE-PGRN cells were also analyzed for secretion of PGRN/PSAP complexes (See sections 12 and 13 for methods of PGRN/PSAP complex monitoring). Encapsulated ARPE-PGRN cells secrete both PGRN and PGRN/PSAP complexes as determined with the aforementioned PGRN and PGRN/PSAP complex assays (
ARPE-19 cells (ATCC®, Manassas, Va.) were grown to 70% confluency in complete media. On the day of transfection, the cell media was discarded and replaced with serum free media. The cells were transfected with a plasmid (Sleeping beauty transposon system) encoding human PSAP cDNA using the Promega® Fugene 6® transfection kit (Promega® Corporation, Madison, Wis.), according to the manufacturer's instructions. Forty-eight (48) hours post transfection, the cells were split 1:10 and plated in 10 cm2 tissue culture dishes in the presence of complete media supplemented with 800 μg/ml Geneticin® (Thermo Fisher Scientific®, Waltham, Mass.). Fourteen (14) days later, individual colonies were harvested and expanded in complete media supplemented with 800 μg/ml Geneticin® (Thermo Fisher Scientific®, Waltham, Mass.). The different clonal ARPE-PSAP cell lines were analyzed for secreted PSAP using an ELISA assay (Thermo Fisher Scientific®, Waltham, Mass.), described in section 11. Encapsulated ARPE-PSAP cells secrete PSAP whereas neither PGRN nor PGRN/PSAP complexes could be detected
ARPE-19-PGRN #56 cells were grown to 70% confluency in complete media and then transfected with a PSAP-encoding plasmid, as described in section 2, with the exception that the PSAP encoding plasmid had the G418 selection gene replaced with a hygromycin selection gene. Generation of clonal cell lines were accomplished as above, besides that both Geneticin® (Thermo Fisher Scientific®, Waltham, Mass.) (800 μg/ml) and Hygromycin™ (Sigma-Aldrich®, St. Louis, Mo.) (500 μg/ml) were included in the media. Clonal cell lines expressing both PGRN and PSAP were identified with ELISA (Thermo Fisher Scientific®, Waltham, Mass.). Encapsulated ARPE-PGRN+PSAP cells secrete mostly PGRN/PSAP complexes as assessed by analysis by of different fractions derived from size exclusion chromatography of conditioned media from ARPE-PGRN+PSAP cells
ARPE-19 cells (ATCC®, Manassas, Va.) were grown to 70% confluency in complete media. On the day of transfection, the cell media was discarded and replaced with serum free media. The cells were transfected with a plasmid (Sleeping beauty transposon system) encoding the construct (signalpeptide-scFv81-Flag-His)3):
using the Promega® Fugene 6® transfection kit (Promega® Corporation, Madison, Wis.), according to the manufacturer's instructions. Forty-eight (48) hours post transfection, the cells were split 1:10 and plated in 10 cm2 tissue culture dishes in the presence of complete media supplemented with 800 μg/ml Geneticin® (Thermo Fisher Scientific®, Waltham, Mass.). Fourteen (14) days later, individual colonies were harvested and expanded in complete media supplemented with 800 μg/ml Geneticin® (Thermo Fisher Scientific®, Waltham, Mass.) (
ARPE-19 cells (ATCC®, Manassas, Va.) were grown to 70% confluency in complete media. At day of transfection, the cell media was discarded and replaced with serum free media. The cells were transfected with a plasmid (Sleeping beauty transposon system) encoding the construct (signalpeptide-scFv49-Flag-His)4):
using the Promega® Fugene 6® transfection kit (Promega® Corporation, Madison, Wis.), according to the manufacturer's instructions. Forty-eight (48) hours post transfection, the cells were split 1:10 and plated in 10 cm2 tissue culture dishes in the presence of complete media supplemented with 800 μg/ml Geneticin® (Thermo Fisher Scientific®, Waltham, Mass.). Fourteen (14) days later, individual colonies were harvested and expanded in complete media supplemented with 800 μg/ml Geneticin® (Thermo Fisher Scientific®, Waltham, Mass.). Expression and secretion of scFv49 was observed (
ARPE-19 cells (ATCC®, Manassas, Va.) were grown to 70% confluency in complete media. On the day of transfection, the cell media was discarded and replaced with serum-free media. The cells were transfected with a plasmid (Sleeping beauty transposon system) encoding the construct (signalpeptide-scFv113-Flag-His)5):
using the Promega® Fugene 6® transfection kit (Promega® Corporation, Madison, Wis.), according to the manufacturer's instructions. Forty-eight (48) hours post transfection, the cells were split 1:10 and plated in 10 cm2 tissue culture dishes in the presence of complete media supplemented with 800 μg/ml Geneticin® (Thermo Fisher Scientific®, Waltham, Mass.). Fourteen (14) days later, individual colonies were harvested and expanded in complete media supplemented with 800 μg/ml Geneticin® (Thermo Fisher Scientific®, Waltham, Mass.) (
Clonal ARPE-PGRN cells were grown to 70% confluency in complete media and then transfected with a PSAP encoding plasmid, as described in section 3, and a plasmid (Sleeping beauty transposon system) encoding the construct (signalpeptide-scFv81-Flag-His) described in section 5. Generation of clonal cell lines were accomplished by culturing the transfectants in presence of Geneticin® (Thermo Fisher Scientific®, Waltham, Mass.) (800 μg/ml) and Hygromycin™ (Sigma-Aldrich®, St. Louis, Mo.) (500 μg/ml). Clonal cell lines expressing PGRN, PSAP and scFv81 were identified with immunocytochemical analysis (ICC). Clone D5 (#D5), expressing PGRN, PSAP and scFv81 was selected for further analysis (
ARPE-19 cells (ATCC®, Manassas, Va.) were grown to 70% confluency in complete media. On the day of transfection, the cell media was discarded and replaced with serum free media. The cells were transfected with the plasmid described in section 3 (Sleeping beauty transposon system, Hygromycin™ (Sigma-Aldrich®, St. Louis, Mo.) selection gene) with a cDNA encoding the following peptide (signalpeptide-Fc-scFv81)6):
using the Promega® Fugene 6® transfection kit (Promega® Corporation, Madison, Wis.), according to the manufacturer's instructions. Forty-eight (48) hours post transfection, the cells were split 1:10 and plated in 10 cm2 tissue culture dishes in the presence of complete media supplemented with 500 μg/ml Hygromycin™ (Sigma-Aldrich®, St. Louis, Mo.). Fourteen (14) days later, individual colonies were harvested and expanded in complete media supplemented with 500 μg/ml Hygromycin™ (Sigma-Aldrich®, St. Louis, Mo.).
ARPE-19 cells (ATCC®, Manassas, Va.) were grown to 70% confluency in complete media. On the day of transfection, the cell media was discarded and replaced with serum free media. The cells were transfected with the plasmid described in section 3 (Sleeping beauty transposon system, Hygromycin™ (Sigma-Aldrich®, St. Louis, Mo.) selection gene) with a cDNA encoding the following peptide (signalpeptide-Fc-scFv49)7):
using the Promega® Fugene 6® transfection kit (Promega® Corporation, Madison, Wis.), according to the manufacturer's instructions. Forty-eight (48) hours post transfection, the cells were split 1:10 and plated in 10 cm2 tissue culture dishes in the presence of complete media supplemented with 500 μg/ml Hygromycin™ (Sigma-Aldrich®, St. Louis, Mo.). Fourteen (14) days later, individual colonies were harvested and expanded in complete media supplemented with 500 μg/ml Hygromycin™ (Sigma-Aldrich®, St. Louis, Mo.) Expression and secretion of Fc-scFv49 was observed (
ARPE-19 cells (ATCC®, Manassas, Va.) were grown to 70% confluency in complete media. On the day of transfection, the cell media was discarded and replaced with serum free media. The cells were transfected with the plasmid described in section 3 (Sleeping beauty transposon system, Hygromycin™ (Sigma-Aldrich®, St. Louis, Mo.) selection gene) with a cDNA encoding the following peptide (signalpeptide-Fc-scFv113)8):
using the Promega® Fugene 6® transfection kit (Promega® Corporation, Madison, Wis.), according to the manufacturer's instructions. Forty-eight (48) hours post transfection, the cells were split 1:10 and plated in 10 cm2 tissue culture dishes in the presence of complete media supplemented with 500 μg/ml Hygromycin™ (Sigma-Aldrich®, St. Louis, Mo.). Fourteen (14) days later, individual colonies were harvested and expanded in complete media supplemented with 500 μg/ml Hygromycin™ (Sigma-Aldrich®, St. Louis, Mo.).
ARPE-19-GDNF cells were grown to 70% confluency in complete media and then transfected with a PSAP-encoding plasmid, as described in section 2, with the exception that the PSAP-encoding plasmid has the G4 1 8 selection gene replaced with a hygromycin selection gene. Generation of clonal cell lines was accomplished as discussed previously, besides that both Geneticin® (Thermo Fisher Scientific®, Waltham, Mass. )(800 μg/ml) and Hygromycin™ (Sigma-Aldrich®, St. Louis, Mo.)(500 μg/ml) were included in the media. Clonal cell lines expressing both GDNF and PSAP were identified with ELISA (Thermo Fisher Scientific®, Waltham, Mass.) as illustrated in
The capture antibody (mouse mAb Abnova® cat no. H00005660-M01, 0.43 mg/ml (Abnova® GmbH, Taipei, TW)) was diluted 1:500 in phosphate buffered saline (PBS) (HyClone® Laboratories, Inc., South Logan, Utah) and 50 μI/well were added to Nunc MaxiSorp™ plates (cat no. 442404 (Thermo Fisher Scientific®, Waltham, Mass.)). The plates were incubated at room temp (RT) overnight (ON). The reaction mixture was then discarded and the wells were subsequently washed three (3) times in PBS/Tween20 (0.1%) (Sigma-Aldrich®, St. Louis, Mo.) prior to the addition of 150 μl blocking solution (PBS/Tween/BSA (2%)) (Alfa Aesar®, Tewksbury, Mass.) to each well. After a two (2) hour incubation at RT, the plates were washed twice in PBS/Tween20 (0.1%) (Sigma-Aldrich®, St. Louis, Mo.) prior to the addition of samples that were diluted in PBS/Tween20 (0.1%)/1% Bis(trimethylsilyl)acetamide (BSA) (Me3SiNC(OSiMe3)Me) (Alfa Aesar®, Tewksbury, Mass.). The binding reactions were left for two (2) hours at RT and then removed, followed by three (3) washes in PBS/Tween20 (0.1%) (Sigma-Aldrich®, St. Louis, Mo.). Then, 50 μl/well of detection antibody (Rb anti-PSAP, HPA004426, 0.1 mg/ml, diluted 1:300 in PBS/Tween20 (0.1%)/BSA (1%) (Alfa Aesar®, Tewksbury, Mass.)) were added and the reactions left for two (2) hours at RT. After another three (3) washes, 50 μl/well of horseradish peroxidase (HRP)-conjugated anti-Rb IgG antibody were added and the plates were incubated for one (1) hour at RT. Finally, the plates were washed three (3) times in PBS/Tween20 (0.1%) (Sigma-Aldrich®, St. Louis, Mo.) and HRP activity monitored using 1 Step™ TMB Ultra reagent (Thermo Fisher Scientific®, Waltham, Mass.) followed by addition of one (1) volume 2M H2SO4 to stop the reactions. Absorbance at 450 nm was subsequently monitored using a Molecular Devices® microplate reader (Promega® Corporation, Madison, Wis.). Human recombinant PSAP was used as standard (ABCAM®, cat nr: 203534 (ABCAM® PLC Co., Cambridge, UK)).
The capture antibody, anti-PGRN antibody (hPGRN ELISA DuoSet® kit (R&D Systems®, Minneapolis, Minn.)) was diluted according to the manufacturer's instructions and 50 μl/well were added in Nunc MaxiSorp™ 96-well plates (cat no. 442404 (Thermo Fisher Scientific®, Waltham, Mass.)). The reactions were incubated on at room temperature (RT). The reactions were then discarded and the plates washed three (3) times in PBS/Tween20 (0.1%) (Sigma-Aldrich®, St. Louis, Mo.). Approximately, 150 μl blocking solution (PBS/Tween/BSA (2%) (Alfa Aesar®, Tewksbury, Mass.)) were subsequently added and the plates incubated for two (2) hours at RT. The plate was then washed twice in PBS/Tween20 (0.1%) (Sigma-Aldrich®, St. Louis, Mo.) prior to addition of samples that were diluted in PBS/Tween20 (0.1%)/BSA (1%) (Alfa Aesar®, Tewksbury, Mass.). After two (2) hours of incubation at RT the plates were washed three (3) times prior to addition of an antibody recognizing PSAP (anti-PSAP, HPA004426, 0.1 mg/ml, diluted 1:300 in PBS/Tween20 (0.1%)/BSA (1%) (Alfa Aesar®, Tewksbury, Mass.)). Reactions were incubated for two (2) hours at RT. After three (3) washes, a horseradish peroxidase (HRP)-conjugated anti-Rb IgG antibody was added and the reactions incubated for one (1) hour at RT. Finally, the plates were washed three (3) times and HRP activity was monitored using 1 Step™ TMB Ultra reagent (Thermo Fisher Scientific®, Waltham, Mass.) followed by addition of 1 volume 2M H2SO4 to stop the reactions. Absorbance was finally read at 450 nm. As standard, purified PGRN/PSAP complexes derived from conditioned media from ARPE-PGRN/PSAP cells were used. PGRN/PSAP was diluted in PBS/Tween20 (0.1%)/BSA (1%) (Alfa Aesar®, Tewksbury, Mass.). As evidenced by
The capture antibody, mouse mAb anti-PSAP antibody (Abnova® cat no. H00005660-M01, 0.43 mg/ml (Abnova® GmbH, Taipei, TW)), was diluted 1:500 in PBS (HyClone® Laboratories, Inc., South Logan, Utah) and 50 μl/well Nunc MaxiSorp™ 96-well plates (cat no. 442404 (Thermo Fisher Scientific®, Waltham, Mass.)) were added. After incubating the plates at room temperature (RT), the reaction mixture was discarded and the plates were washed three (3) times in PBS/Tween20 (0.1%) (Sigma-Aldrich®, St. Louis, Mo.). Then, 150 μl blocking solution (PBS/Tween/BSA (2%) (Alfa Aesar®, Tewksbury, Mass.)) was added and the plates incubated for two (2) hours at room temp. The plates were then washed twice in PBS/Tween20 (0.1%) (Sigma-Aldrich®, St. Louis, Mo.) prior to addition of samples, which were diluted in PBS/Tween20 (0.1%)/BSA (1%) (Alfa Aesar®, Tewksbury, Mass.). The reactions were left for two (2) hours at RT and the removed, followed by three (3) washes in PBS/Tween20 (0.1%) (Sigma-Aldrich®, St. Louis, Mo.). Then, 50 μl/well of detection antibody (biotinylated anti-PGRN antibody, R&D Systems® hPGRN ELISA DuoSet® kit (R&D Systems®, Minneapolis, Minn.), diluted according to the manufacturer's instructions in PBS/Tween20 (0.1%)/BSA (1%)(Alfa Aesar®, Tewksbury, Mass.)) were added and the reactions left for two (2) hours at RT. After another three (3) washes, 50 μl/well of HRP-conjugated Streptavidin (Thermo Fisher Scientific®, Waltham, Mass.) were added and the plates incubated for thirty (30) minutes at RT. Finally, the plates were washed 3 times and HRP activity monitored using 1 Step™ TMB Ultra reagent (Thermo Fisher Scientific®, Waltham, Mass.) followed by the addition of one (1) volume of 2M H2SO4 to stop the reactions. Absorbance was finally read at 450 nm.
The aforementioned cell lines were encapsulated according to a method previously described; from herein the encapsulated cell lines are denoted PGRN-, PSAP- and PGRN+PSAP devices, respectively. All devices were cultured in Gibco HE-SFM medium (Thermo Fisher Scientific®, Waltham, Mass.) at 37° C., 5% CO2 for extended time periods, ranging from two (2) weeks to five (5) months. Aliquots of conditioned medium were analyzed for PGRN, PSAP and PGRN+PSAP secretion. PGRN-devices secrete both PGRN and PGRN/PSAP complexes, PSAP-devices secrete only PSAP and PGRN+PSAP devices secrete only PGRN/PSAP complexes (
Cells were grown to confluency in 225 cm2 tissue culture dishes in complete media. The cell media was then replaced with Gibco® FreeStyle™ 293 Expression Medium (Invitrogen/Thermo Fisher Scientific®, Carlsbad, Calif.) and the cells were cultured for seventy-two (72) hours at 37° C., 5% CO2. Media was subsequently recovered and concentrated using Amicon® Ultra-4 Centrifugal filters (Millipore Sigma®, Burlington, Mass.) cut-off 30 kDa. Mouse primary cortical neurons, prepared from embryonic day seventeen (17) and cultured for twelve to fourteen (12-14) days (Div 12-14) at 37° C., 5% CO2, were exposed to the concentrated conditioned media so the final [PGRN], [PSAP] or [PGRN/PSAP] was 1 μg/ml. The reactions were left overnight and the cells were subsequently fixed and subjected to immunocytochemical analysis using antibodies specific for different lysosomal markers, including PGRN, PSAP and LAMP1.
The different ARPE-cell lines were grown to confluency in 225 cm2 tissue culture dishes in complete media. The cell media was then replaced with Gibco® FreeStyle™ 293 Expression Medium (Invitrogen/Thermo Fisher Scientific®, Carlsbad, Calif.) and the cells were cultured for another seventy-two (72) hours at 37° C., 5% CO2. The conditioned media was then recovered and concentrated using Amicon® Ultra-4 Centrifugal filters (Millipore Sigma®, Burlington, Mass.) cut-off 30 kDa. Primary mouse cortical neurons, Div 12-14, were subsequently exposed to the concentrated conditioned media at 1 μg/ml final [PGRN] and [PGRN/PSAP]. The reactions were left for twenty (24) hours at 37° C., 5% CO2, and then terminated by discarding the media from the Nunc MaxiSorp™ 96-well plates (cat no. 442404 (Thermo Fisher Scientific®, Waltham, Mass.)) followed by addition of activity buffer NaCitrate™ ((trisodium citrate dihydrate) (Sigma-Aldrich®, St. Louis, Mo.) (pH 5.4)), Triton™ X-100 (Sigma-Aldrich®, St. Louis, Mo.) (0.25% (v/v)), Taurocholic acid (2-{[(3α,5β,7α,12α)-3,7,12-trihydroxy-24-oxocholan-24-yl]amino}ethanesulfonic acid) (Sigma-Aldrich®, St. Louis, Mo.) (0.25% (w/v)) and 1 mM EDTA (2,2′,2″,2″′-(Ethane-1,2-diyldinitrilo)tetraacetic acid) (Sigma-Aldrich®, St. Louis, Mo.) after which the plates were immediately put in −85° C. to allow efficient lysis of the cells. To monitor GBA1 activity, the lysates were first thawed and incubated on ice for twenty (20) minutes before centrifugation at 4° C. for twenty (20) minutes at 20000 RCF to remove cell debris. The supernatants were collected and divided into two aliquots to test for GBA1 activity and to determine protein concentration, respectively. To test for GBA1 activity, the lysates were mixed with 1% BSA (Alfa Aesar®, Tewksbury, Mass.), 1 mM 4-Methylumbelliferyl b-glucophyranoside ((4-MU) (#M3633) (Sigma-Aldrich®, St. Louis, Mo.)) in 50 μl volume and then incubated at 37° C. for forty (40) minutes. The reactions were stopped with 1 volume of 1 M glycine (Sigma-Aldrich®, St. Louis, Mo.), pH 12.5, and the fluorescence monitored (ex=355 nm, em=460 nm) using a SpectraMax® D5 Series Multi-Mode Microplate Reader (Molecular Devices®, San Jose, Calif.).
The in vivo functionality of the devices was tested by striatal implantation in rats in a manner similar to the study outline previously described (Tornøe J et al., (2012), Restor Neurol Neurosci, 30(3):225-36). Rats were treated for four to twenty-four (4 to 24) weeks and the devices were then removed for functionality testing by monitoring their PGRN, PSAP and PGRN/PSAP complex release.
Sprague Dawley® native rats (Charles River Laboratories, Wilmington, Mass.), treated for twenty-four (24) weeks with PGRN-devices, were sacrificed and the brains recovered and subjected to fixation and paraffin embedding (ABCAM® PLC Co., Cambridge, UK) for histopathological assessment. Coronal sections (5 um) were incubated with antibodies raised against human PGRN, Ki67, GFAP, Iba1 and CD3 to monitor exposure, proliferating cells, inflammatory reactions and infiltrating T-cells, respectively. As shown in
PGRN-, PSAP- and PGRN+PSAP-scFv81 devices were implanted in the striatum of rats that also got an injection in substantia nigra of AAV9 virus carrying a human alpha-synuclein gene (Decressac M et al., (2012), Neurobio Dis, 45(3):939-953). The rats were subjected to behavioral testing four, eight and twelve (4, 8, and 12) weeks post device implantation/virus injection. The rats were then sacrificed and the brains recovered for histopathological assessment (
PGRN-, PSAP- and PGRN+PSAP-devices were implanted in the striatum of rats as described before (Tornøe J et al., (2012), Restor Neurol Neurosci, 30(3):225-36). A week post-surgery, the rats were subjected to behavioral testing (
GDNF+PGRN secreting devices, GDNF+PSAP secreting devices and GDNF+PGRN+PSAP secreting devices show therapeutic activity in the rat 6-OHDA model of neurodegeneration. Devices filled with ARPE-GDNF and either of the ARPE-factor cell lines described in Example 12 above, were tested for therapeutic activity as described in Example 12. All treatments showed therapeutic activity (
ARPE-19-PGRN+PSAP clone #D5 cells were cultured in complete media. Three flasks (225 cm2) were trypsinated (TrypLE™ Express Enzyme, Gibco®, 12605-010 (Thermo Fisher Scientific®, Waltham, Mass.)) and the cells were resuspended in 550 ml complete media and then seeded in a Corning® HYPERFlask® M Cell Culture Vessel (1720 cm2 area)(Sigma-Aldrich®, St. Louis, Mo.). Three (3) days post seeding, the media was removed and the cells were washed with 2×100 ml PBS (HyClone® Laboratories, Inc., South Logan, Utah). Then, 550 ml Gibco® FreeStyle™ 293 Expression Media (Thermo Fisher Scientific®, Waltham, Mass.) supplemented with 1x Gibco® Penicillin-Streptomycin (PEST) (Thermo Fisher Scientific®, Waltham, Mass.), Geneticin® (G418) (Life Technologies® Corporation, Carlsbad, Calif.) and Hygromycin™ (Sigma-Aldrich®, St. Louis, Mo.) were added. The cells were cultured for ninety-six (96) hours at 37° C., 5% CO2. The conditioned media was collected and replaced with 550 ml fresh Gibco® FreeStyle™ 293 Expression Media (Thermo Fisher Scientific®, Waltham, Mass.), supplemented with 1x Gibco® Penicillin-Streptomycin (PEST) (Thermo Fisher Scientific®, Waltham, Mass.), Geneticin® (G418) (Life Technologies® Corporation, Carlsbad, Calif.) and Hygromycin™ (Sigma-Aldrich®, St. Louis, Mo.). The collected conditioned media was immediately frozen at −85° C. for later further protein purification.
Frozen batches of cell culture media were slowly thawed overnight at 4° C. The media was then centrifuged at 7200 rcf for twenty (20) minutes to pellet dead cells and debris. To secure the complete removal of particles, the supernatant was sterile filtered/degassed using Sarstedt® Filtration Units (0.22 um filters, ref 83.3941.101)(Sarstedt® AG & Co., Nuembrecht, Del.) prior to further processing. Subsequently, the filtered conditioned media was concentrated ten (10) times using Amicon® Cell Filter stir technology (cat no. UFSC40001) (Millipore Sigma®, Burlington, Mass.) loaded with a 30 kDa cut-off filter. The resulting concentrate was then subjected to different chromatographic methods.
Two different chromatographic methods to purify PGRN/PSAP complexes were applied: ion exchange chromatography and size exclusion chromatography (SEC), respectively. First, the concentrated sterile filtered media was run on a 6 ml Diethylaminoethyl cellulose (DEAE) column (Waters™ Technology Corporation, Milford, Mass.) and eluted with a 0-50% gradient 2M NaCl solution. Fractions were subsequently analyzed with SDS-PAGE gel (Thermo Fisher Scientific®, Waltham, Mass.) and PGRN/PSAP complex assay. PGRN/PSAP complexes were identified in three (3) fractions that were pooled and further subjected for SEC.
To ensure efficient separation, a HiLoad® 26/60 Superdex® 200 PG column (GE Healthcare Process R&D AB, Uppsala, SE) was used. Fractions containing PGRN/PSAP complexes were identified using three (3) different assays: PSAP ELISA (& Example 5), PGRN ELISA (hPGRN ELISA DuoSet® kit (#DY2420) (R&D Systems®, Minneapolis, Minn.)) and a PGRN/PSAP assay (& Example 5). Aliquots from each fraction were analyzed by these assays as well as by SDS-PAGE analysis (Thermo Fisher Scientific®, Waltham, Mass.). PGRN/PSAP complexes eluate in fractions different from free PGRN (
The pooled fractions with PGRN/PSAP complexes were dialyzed overnight in sterile PBS solution (HyClone® Laboratories, Inc., South Logan, Utah), aliquoted in sterile polypropylene Eppendorf® Tubes (Eppendorf® AG, Hamburg, Del.), snap frozen, and stored at −85° C.
Extracellularly administered PGRN/PSAP interacts efficiently with mouse cortical primary neurons, is internalized and targets the lysosome. Mouse primary cortical neurons, prepared from embryonic day seventeen (17) were cultured at 37° C., 5% CO2 for fourteen (14) days in BD Falcon™ 96-well cell culture dishes (BD Biosciences, Bedford, Mass.) prior to treatment. Sampled of PGRN, PSAP or PGRN/PSAP concentrated to 1 to 5 μg/ml were then added and the cultures incubated at 37° C., 5% CO2. The media was subsequently removed and the cells fixed and subjected to immunocytochemical analysis using antibodies specific for different lysosomal markers, including PGRN, PSAP and GBA1. As evidenced by
An extracellularly administered PGRN/PSAP complex, PGRN or PSAP colocalize with, and each treatment activate, GBA1 in mouse primary cortical neurons and human primary fibroblasts, derived from heterozygous GBA1 L444P mutation carriers. Mouse primary cortical neurons prepared from embryonic day seventeen (17) were cultured at 37° C., 5% CO2 for fourteen (14) days in BD Falcon™ 96-well cell culture dishes (BD Biosciences, Bedford, Mass.) prior to treatment. PGRN, PSAP or PGRN/PSAP were then added to a concentration of 10 ng/ml and cultures were incubated for twenty-four (24) hours prior to analysis. Human fibroblasts were grown to confluency and then treated with PGRN, PSAP or PGRN/PSAP GBA1 as aforementioned described for the mouse primary neuronal cultures. After twenty-four (24) hours of treatment at 37° C., 5% CO2, the reactions were terminated by removing the media from the Nunc MaxiSorp™ 96-well plates and adding an activity buffer consisting of NaCitrate™ ((trisodium citrate dihydrate) (pH 5.4) (Sigma-Aldrich®, St. Louis, Mo.)), Triton™ X-100 ((0.25% (v/v)) (Sigma-Aldrich®, St. Louis, Mo.)), taurocholic acid ((2-{[(3α,5β,7α,12α)-3,7,12-trihydroxy-24-oxocholan-24-yl]amino}ethanesulfonic acid) (0.25% (w/v) (Sigma-Aldrich®, St. Louis, Mo.)) and 1 mM EDTA ((2,2′,2″,2″′-(Ethane-1,2-diyldinitrilo)tetraacetic acid) (Sigma-Aldrich®, St. Louis, Mo.)) were added and the plates were put in −85° C. to allow efficient lysis and brake of cells. To monitor GBA1 activity, the lysates were first thawed and incubated on ice for twenty (20) minutes before centrifugation at 4° C. for twenty (20) minutes at 20000 rcf to remove cell debris. The supernatants were collected and divided into two (2) aliquots for GBA1 activity and protein concentration determination, respectively. For GBA1 activity, the lysates were mixed with 1% BSA (Alfa Aesar®, Tewksbury, Mass.), 1 mM 4-Methylumbelliferyl b-glucophyranoside (4-MU, Sigma-Aldrich®, #M3633) in 50 μl volume and then incubated at 37° C. for forty (40) minutes. The reactions were stopped with one (1) volume of 1 M glycine at pH 12.5 and the fluorescence was monitored (ex=355 nm, em=460 nm) using a Spectramax® D5 plate reader (Molecular Devices®, San Jose, Calif.).
Extracellularly administered PGRN/PSAP stimulates neurite outgrowth in mouse primary cortical neuronal cultures. Mouse primary cortical neurons were prepared from embryonic day seventeen (17). Brains were harvested and cortical cultures prepared according to known methods. (Merino-Serrais P et al., (2019), Cereb Cortex, 29(1):429-46). Cells were seeded in BD Falcon™ 96-well Poly-L-coated cell culture dishes (BD Falcon™ 96-well cell culture dishes (BD Biosciences, Bedford, Mass.)) in neurobasal media (Neurobasal™ Plus Medium, (Gibco®, Life Technologies®, Carlsbad, Calif.), supplemented with L-Glutamine, Gibco® Penicillin-Streptomycin (PEST) (Thermo Fisher Scientific®, Waltham, Mass.) and 2% B27 (Anti-Human Leukocyte Antigen B27 antibody (ABCAM® PLC Co., Cambridge, UK). Six (6) hours post seeding, the media was removed and directly replaced with 90 μl/well neurobasal media (Neurobasal™ Plus Medium, (Gibco®, Life Technologies®, Carlsbad, Calif.)), supplemented with L-Glutamine, Gibco® Penicillin-Streptomycin (PEST) (Thermo Fisher Scientific®, Waltham, Mass.) and either 0%, 0.5 or 1% B27 (Anti-Human Leukocyte Antigen B27 antibody (ABCAM® PLC Co., Cambridge, UK). Cultures with complete media, i.e. 2% B27 (Anti-Human Leukocyte Antigen B27 antibody (ABCAM® PLC Co., Cambridge, UK), served as control. Ten (10) μl neurobasal media (Neurobasal™ Plus Medium, (Gibco®, Life Technologies®, Carlsbad, Calif.)), supplemented with L-Glutamine and Gibco® Penicillin-Streptomycin (PEST) (Thermo Fisher Scientific®, Waltham, Mass.) and 10 ng/ml of either PGRN or PGRN/PSAP were then added and the cultures were further incubated at 37° C. for four (4) days or, approximately ninety-six (96) hours. Media was then discarded and the cells fixed in 4% formaldehyde (O═CH2) (Fisher Chemical, Waltham, Mass.) for thirty (30) minutes at room temp. The fixation solution was subsequently discarded and the wells washed three (3) times with PBS (HyClone® Laboratories, Inc., South Logan, Utah) prior to immunocytochemical (ICC) analysis: fixed cells were first treated for one (1) hour at room temperature with PBS (HyClone® Laboratories, Inc., South Logan, Utah)/ Triton™ X-100 (t-Oct-C6H4-(OCH2CH2)xOH, x=9-10, (MilliporeSigma®, Burlington, Mass.)) (0.25%) and BSA (Alfa Aesar®, Tewksbury, Mass.) (3%) to permeabilize the cells and to block nonspecific protein binding. After removing the blocking solution, the cultures were exposed to fresh blocking solution supplemented with mouse monoclonal anti-Tubulin antibodies ((anta Cruz, G8) (ABCAM® PLC Co., Cambridge, UK)), diluted to 1:200, and then incubated on at +4° C. Subsequently, cells were washed three (3) times in with PBS (HyClone® Laboratories, Inc., South Logan, Utah)/ Triton™ X-100 (MilliporeSigma®, Burlington, Mass.) (0.25%) and BSA (Alfa Aesar®, Tewksbury, Mass.) (3%) and then incubated for one (1) hour at room temperature with a blocking solution supplemented with Alexa594 goat anti-mouse IgG (1:1000) (Alexa Fluor™, Thermo Fisher Scientific®, Waltham, Mass.) and 10 ug/ul bisBenzimide blue fluorescent dye, i.e. Hoechst stain (Höchst, Frankfurt, Del.). After removal of the reaction mixture, the BD Falcon™ 96-well plates were washed three (3) times in PBS/Triton™ x-100, i.e. PBS (HyClone® Laboratories, Inc., South Logan, Utah)/ Triton X-100 (MilliporeSigma®, Burlington, Mass.) and then stored in a dark environment at +4° C. prior to analysis. Neuronal morphology was monitored using a High-Content Screening Platform (HCS) part of Cellomics™ technology (ArrayScan™, Thermo Fisher Scientific®, Waltham, Mass.) and the neurite morphology software (Data61®, CSIRO, Canberra, AU). PGRN/PSAP, like PGRN, stimulates neurite outgrowth as shown in
Conditioned media from ARPE-PGRN and ARPE-PSAP cells were prepared, concentrated and buffer exchanged to PBS. Recombinant PGRN-His (cat no. 2420-PG (R&D Systems®, Minneapolis, Minn.)) was mixed with the concentrated conditioned media from ARPE-PSAP cells and PGRN/PSAP complexes were formed as demonstrated by western blot analysis
For convenience, certain terms employed in the specification, examples and appended claims are collected here. These definitions should be read in light of the disclosure and understood by a person of ordinary skill in the art.
As used herein, the term “bioreactor” refers to any manufactured device or system that supports a biologically active environment. In various embodiments, a bioreactor is a vessel in which a chemical process is carried out which involves organisms or biochemically active substances derived from such organisms. This process can either be aerobic or anaerobic. In a further embodiment, a bioreactor may also refer to a device or system designed to grow cells or tissues in the context of cell culture. In yet a further embodiment, molecules secreted or produced by the cells grown in a bioreactor may be harvested and purified.
As used herein, the terms “capsule,” or alternatively, “encapsulate” or “encapsulated,” refer to an enclosed device (or method of using said device) containing cells, preferably by a semi-permeable membrane that permits the bidirectional diffusion of molecules such as the influx of oxygen, nutrients, growth factors etc., essential for cell metabolism and the outward diffusion of waste products and therapeutic proteins. At the same time, the semi-permeable nature of the membrane prevents immune cells and antibodies from destroying the encapsulated cells regarding them as foreign invaders.
As used herein, the term “cell line” refers to a population of cells derived from a single progenitor cell that can be propagated repeatedly or indefinitely. The progenitor cell may be derived from the organ or tissue of a larger animal or plant.
As used herein, the terms “expression,” or alternatively, “express, ” “expressing, ” “expressed” or “to express, ” refer to the transcription and stable accumulation of sense RNA (mRNA) or antisense RNA derived from a nucleic acid or polynucleotide. Expression may also refer to translation of mRNA into a protein or polypeptide.
As used herein, the term “expression construct” refers to any molecule, virus, or organism designed to introduce a nucleic acid or polynucleotide into a cell for the purpose of expressing a protein or RNA encoded by that nucleic acid or polynucleotide. In a preferred embodiment, an expression construct may be a plasmid. An expression construct may also refer to an expression vector, and these terms are used interchangeably.
As used herein, the terms “fragment,” or alternatively, “a fragment thereof,” when applied to a polynucleotide sequence refer to a nucleotide sequence comprising the same nucleotide sequence as the reference nucleic acid over a common portion with a reduced length relative to the reference nucleic acid. Such a nucleic acid fragment according to the invention may be contained in a larger polynucleotide, if appropriate, which is a constituent thereof.
As used herein, the terms “heterodimer,” or alternatively, “heterodimers” or “heterodimerization,” refer to a macromolecular complex formed by two protein monomers, or single proteins, wherein the two protein monomers comprise two different protein sequences.
As used herein, the term “immunoisolatory” refers to a method or means of protecting implanted material such as biopolymers, cells, or drug release carriers from an immune reaction or minimizing an immune reaction. In an embodiment, an implantable device may be immunoisolatory in that in protects material inside of the device from an immune reaction after the device is implanted in a host.
As used herein, the terms “implantable,” or alternatively, “implant,” “implants,” “implanted,” or “to implant, ” refer to a device designed to be introduced into the body of a host for an extended period of time, for the purpose of replacing, augmenting, or supporting an existing biological structure or function of the host.
As used herein, the term “matrix” refers to a three-dimensional network of extracellular macromolecules, such as polymers, collagen, enzymes, laminin, fibronectin, or glycoproteins, that provide structural and biochemical support to surrounding cells.
As used herein, the terms “modify,” or alternatively, “modified,” “modifies,” “modification,” “modifying” or “to modify, ” refer to any alteration of matter which, directly or indirectly, enhances, diminishes, adds, or removes a property or properties of said matter.
As used herein, the terms “neurological disease” and “neurological disorder” are used interchangeably and refer to any functional abnormality or disturbance of the nervous system, whether caused by structural, biochemical, or electrical abnormalities in the brain, spinal cord or other nerves.
As used herein, the term “other chromatographic methods” refers to any technique used for the separation of a mixture, whether preparative or analytical, as known in the art at the time of filing or discovered thereafter.
As used herein, the terms “precursor polypeptide,” “protein precursor,” or “pro-protein” are used interchangeably and refer to an inactive protein (or peptide) that can be turned into an active form by post-translational modification, such as breaking off a piece of the molecule or adding on another molecule.
As used herein, the terms “purified,” or alternatively, “purify,” “purified,” “purification” or “to purify” refer to a substance which has been substantially increased in concentration or freed of contaminants. This term does not necessarily indicate absolute purity unless otherwise indicated.
As used herein, the term “Sleeping beauty Transposase System” refers to a method of introducing DNA sequences into the genome of a cell by means of a Sleeping beauty transposase and a transposon, as well as materials to perform said method.
As used herein, the terms “subpeptide,” or alternatively, “subpeptides” or “subpeptides thereof” refer to a polypeptide that is derived from part of a larger protein or polypeptide. In an embodiment, the subpeptide may be a fragment of the larger protein or polypeptide.
As used herein, the terms “therapeutic,” or alternatively, “a therapeutic,” “a therapeutic drug,” “a therapeutic agent,” “therapy,” “therapies,” “a therapeutic regimen” or “a therapeutic method” refer to any molecule (or method using said molecule) that confers a beneficial function to the subject being treated with said molecule. Therapeutics may include, but are not limited to, peptides, polypeptides, single or multi-chain proteins, fusion proteins, antisense oligonucleotides, small interfering RNAs, ribozymes, and RNA external guide sequences. The therapeutic may include naturally occurring sequences, synthetic sequences, or a combination of natural and synthetic sequences.
As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Further, unless expressly stated to the contrary, ‘or’ refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present). Also, use of the terms “a” or “an” are employed to describe elements and components of the invention. This is done merely for convenience and to give a general sense of the invention. This description should be read to include one or at least one and the singular also includes the plural unless it is obvious that it is meant otherwise. Unless as otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are discussed above. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. In the following description, numerous specific details are provided, such as the identification of various system components, to provide an understanding of embodiments of the invention. One skilled in the art will recognize, however, that embodiments of the invention can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In still other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of various embodiments of the invention Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearance of the phrases “in one embodiment” or “in an embodiment” in various places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
The term “and/or” as used herein is defined as the possibility of having one or the other or both. For example, “A and/or B” provides for the scenarios of having just A or just B or a combination of A and B. If the claim reads “A and/or B and/or C, ” the composition may include A alone, B alone, C alone, A and B but not C, B and C but not A, A and C but not B or all three A, B, and C components.
For convenience, certain terms employed in the specification, examples and appended claims are collected here. These definitions should be read in light of the disclosure and understood as by a person of ordinary skill in the art.
The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.
Unless otherwise indicated, all numbers expressed quantities of ingredients, reaction conditions, and so forth use in the specification and claims are to be understood as being modified in all instances by the term “about,” defined as ±5%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention.
The above discussion is meant to be illustrative of the principle and various embodiments of the present invention. Numerous variations, combinations and modifications will become apparent to those skilled in the art once the above disclosure is fully appreciated. It is intended that the following claims be interpreted to embrace all such variations and modifications.
This invention was not made with U.S. government support.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US21/38312 | 6/21/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63212721 | Jun 2021 | US | |
| 63040925 | Jun 2020 | US |
