Philip Kusk | Uppsala University Campus Gotland (original) (raw)

Papers by Philip Kusk

Journal of Virological Methods, 1991

A novel competition ELBA for detection of antibodies against HIV-1 was developed. The assay is ba... more A novel competition ELBA for detection of antibodies against HIV-1 was developed. The assay is based on competition at the single epitope level and utilises a human monoclonal antibody and an E. co&produced fragment of the transmembrane glycoprotein gp41. The sensitivity of the assay was 100% in tests on 247 serum samples obtained from 219 individuals previously shown to be HIV-l antibody positive by both conventional indirect ELISA and the immunoblotting test. The patients represented various clinical and immunological stages of HIV-1 infection. Likewise, the specificity of the assay was 100% in tests on 105 serum samples from normal individuals previously tested negative by indirect ELISA. Further, among 105 serum samples selected due to consistent false positive reactions in the indirect ELISA only 2 samples (1.9%) demonstrated false positive reactions in the competition ELISA, i.e. 98.1% specificity. Finally, only 2 of 57 (3.5%) serum samples from HIV-2 infected individuals showed positive reactions in the assay, while 54 (94.7%) had absorbance values similar to the negative controls. These results demonstrate that human monoclonal antibodies may form the basis for highly sensitive and specific assays for detection of antibodies to HIV-1.

AIDS, 1992

To map epitopes on gp120 defined by human antibodies and to examine the neutralizing activity of ... more To map epitopes on gp120 defined by human antibodies and to examine the neutralizing activity of these antibodies. Serum from HIV-1-antibody-positive individuals was used to screen a random fragment expression library representing gp120 from the HIVIIIB clone BH10. The library was based on the pUEX1 expression vector. Serum was tested for in vitro neutralizing activity using H9 cells and the HIVIIIB isolate. Four different epitopes defined by human antibodies were mapped on gp120. Two of these have not previously been reported and are located within amino acids (aa) 90-100 in the C1 region and aa 355-365 in the semi-conserved region between V3 and V4. The other two are located within aa 140-145 and aa 286-309. These epitopes are situated in regions that have been shown to demarcate human epitopes. Three serum samples with neutralization titres > or = 1024 were identified. None of the purified antibody fractions defining the mapped epitopes on gp120 had any neutralizing capacity against HIVIIIB. This study is the first demonstration of the applicability of random fragment expression libraries for the direct screening of human serum in order to map epitopes on gp120. Two new epitopes and two previously identified epitopes were mapped in this way. However, none of the linear epitopes was defined by antibody fractions neutralizing HIVIIIB, and it was not possible to map epitopes defined by neutralizing antibodies in the serum samples capable of neutralizing HIVIIIB infection of H9 cells. Thus, it appears that the neutralizing activity of serum in this study was not due to anti-gp120 antibodies defining linear epitopes.

AIDS Research and Human Retroviruses, 1992

Three murine monoclonal antibodies (MAbs) F5-2, F5-4, and F5-16 defining three different epitopes... more Three murine monoclonal antibodies (MAbs) F5-2, F5-4, and F5-16 defining three different epitopes on the major core protein p24 of the human immunodeficiency virus type 1 (HIV-1) were epitope mapped using a random fragment expression library representing the p17- and p24-encoding part of the gag open reading frame. F5-2 defined an epitope within amino acids (aa) 14-23 at the N-terminus of p24, and F5-4 defined an epitope within aa 153-174 in the C-terminus of p24. F5-16 did not recognize any of the fusion proteins produced by the expression library indicating that this MAb defines a true conformational epitope on p24. Since the N-terminus of p24 has been reported to be immunosilent in humans, 356 HIV-1 antibody-positive serum samples were tested for reactivity against the region of p24 defined by F5-2. More than one third of the samples recognized this region indicating that it is immunoreactive and, further, the presence of antibodies against this region was associated with a reduced CD4 cell count.

The Open Tissue Engineering and Regenerative Medicine Journal, 2012

... Lone Fjord-Larsen*,1, Philip Kusk1, Malene Torp1, Jens Christian H. Sørensen2, Kaare Ettrup2,... more ... Lone Fjord-Larsen*,1, Philip Kusk1, Malene Torp1, Jens Christian H. Sørensen2, Kaare Ettrup2, Carsten R. Bjarkam2, Jens Tornøe1, Bengt ... Five female Göttingen minipigs (Ellegaard Göttingen Minipigs ApS, DK), weighing 7.8±0.2 kg at the start of the experiment, were ...

Journal of Bone and Mineral Metabolism, 2004

Experimental Cell Research, 2009

Neural stem cells (NSCs) are powerful research tools for the design and discovery of new approach... more Neural stem cells (NSCs) are powerful research tools for the design and discovery of new approaches to cell therapy in neurodegenerative diseases like Parkinson's disease. Several epigenetic and genetic strategies have been tested for long-term maintenance and expansion of these cells in vitro.

ABSTRACT The present invention relates to the field of therapeutic use of proteins, genes and cel... more ABSTRACT The present invention relates to the field of therapeutic use of proteins, genes and cells, in particular to the therapy based on secreted therapeutic proteins, NsG29 and NsG31. NsG29 and NsG31 are members of a newly identified family of growth factors with a specific cystein pattern and characterised by expression in the nevous system. The secreted growth factors have potential for the treatment of disorders of the nervous system. The invention also relates to bioactive NsG29 and NsG31 polypeptide fragments and the corresponding encoding DNA sequences

Experimental Neurology, 2005

Protein injection studies of the glial cell line derived neurotrophic factor (GDNF) family member... more Protein injection studies of the glial cell line derived neurotrophic factor (GDNF) family member Neurturin (NTN) have demonstrated neuroprotective effects on dopaminergic (DA) neurons, which are selectively lost during Parkinson's disease (PD). However, unlike GDNF, NTN has not previously been applied in PD models using an in vivo gene therapy approach. Difficulties with lentiviral gene delivery of wild type (wt) NTN led us to examine the role of the pre-pro-sequence, and to evaluate different NTN constructs in order to optimize gene therapy with NTN. Results from transfected cultured cells showed that wt NTN was poorly processed, and secreted as a pro-form. A similarly poor processing was found with a chimeric protein consisting of the pre-pro-part from GDNF and mature NTN. Moreover, we found that the biological activity of pro-NTN differs from mature NTN, as pro-NTN did not form a signaling complex with the tyrosine kinase receptor Ret and GFRa2 or GFRa1. Deletion of the pro-region resulted in significantly higher secretion of active NTN, which was further increased when substituting the wt NTN signal peptide with the immunoglobulin heavy-chain signal peptide (IgSP). The enhanced secretion of active mature NTN using the IgSP-NTN construct was reproduced in vivo in lentiviral-transduced rat striatal cells and, unlike wt NTN, enabled efficient neuroprotection of lesioned nigral DA neurons, similar to GDNF. An in vivo gene therapy approach with a modified NTN construct is therefore a possible treatment option for Parkinson's disease that should be further explored. D

Mol Ther, 2010

Nerve growth factor (NGF) prevents cholinergic degeneration in Alzheimer's disease (AD) and impro... more Nerve growth factor (NGF) prevents cholinergic degeneration in Alzheimer's disease (AD) and improves memory in AD animal models. In humans, the safe delivery of therapeutic doses of NGF is challenging. For clinical use, we have therefore developed an encapsulated cell (EC) biodelivery device, capable of local delivery of NGF. The clinical device, named NsG0202, houses an NGF-secreting cell line (NGC-0295), which is derived from a human retinal pigment epithelial (RPE) cell line, stably genetically modified to secrete NGF. Bioactivity and correct processing of NGF was confirmed in vitro. NsG0202 devices were implanted in the basal forebrain of Göttingen minipigs and the function and retrievability were evaluated after 7 weeks, 6 and 12 months. All devices were implanted and retrieved without associated complications. They were physically intact and contained a high number of viable and NGF-producing NGC-0295 cells after explantation. Increased NGF levels were detected in tissue surrounding the devices. The implants were well tolerated as determined by histopathological brain tissue analysis, blood analysis, and general health status of the pigs. The NsG0202 device represents a promising approach for treating the cognitive decline in AD patients.

Epilepsia, 2014

Litsa Nikitidou finished her PhD in Sweden and is currently a postdoctoral researcher in Hungary.

Molecular Therapy, 2010

Nerve growth factor (NGF) prevents cholinergic degeneration in Alzheimer's disease (AD) and impro... more Nerve growth factor (NGF) prevents cholinergic degeneration in Alzheimer's disease (AD) and improves memory in AD animal models. In humans, the safe delivery of therapeutic doses of NGF is challenging. For clinical use, we have therefore developed an encapsulated cell (EC) biodelivery device, capable of local delivery of NGF. The clinical device, named NsG0202, houses an NGF-secreting cell line (NGC-0295), which is derived from a human retinal pigment epithelial (RPE) cell line, stably genetically modified to secrete NGF. Bioactivity and correct processing of NGF was confirmed in vitro. NsG0202 devices were implanted in the basal forebrain of Göttingen minipigs and the function and retrievability were evaluated after 7 weeks, 6 and 12 months. All devices were implanted and retrieved without associated complications. They were physically intact and contained a high number of viable and NGF-producing NGC-0295 cells after explantation. Increased NGF levels were detected in tissue surrounding the devices. The implants were well tolerated as determined by histopathological brain tissue analysis, blood analysis, and general health status of the pigs. The NsG0202 device represents a promising approach for treating the cognitive decline in AD patients.

ABSTRACT Disclosed are NsG28, NsG30, NsG32 polypeptides, nucleic acids encoding NsG28, NsG30, NsG... more ABSTRACT Disclosed are NsG28, NsG30, NsG32 polypeptides, nucleic acids encoding NsG28, NsG30, NsG32 polypeptides, and antibodies that bind to NsG28, NsG30, NsG32 polypeptides as well as methods of making and using the same.

Journal of Neurosurgery, 2012

The authors describe the first clinical trial with encapsulated cell biodelivery (ECB) implants t... more The authors describe the first clinical trial with encapsulated cell biodelivery (ECB) implants that deliver nerve growth factor (NGF) to the cholinergic basal forebrain with the intention of halting the degeneration of cholinergic neurons and the associated cognitive decline in patients with Alzheimer disease (AD). The NsG0202 implant (NsGene A/S) consists of an NGF-producing, genetically engineered human cell line encapsulated behind a semipermeable hollow fiber membrane that allows the influx of nutrients and the efflux of NGF. The centimeter-long capsule is attached to an inert polymer tether that is used to guide the capsule to the target via stereotactic techniques and is anchored to the skull at the bur hole. Six patients with mild to moderate AD were included in this Phase Ib open-label safety study and were divided into 2 dose cohorts. The first cohort of 3 patients received single implants targeting the basal nucleus of Meynert (Ch4 region) bilaterally (2 implants per patient), and after a safety evaluation, a second cohort of 3 patients received bilateral implants (a total of 4 implants per patient) targeting both the Ch4 region and the vertical limb of the diagonal band of Broca (Ch2 region). Stereotactic implantation of the devices was successfully accomplished in all patients. Despite extensive brain atrophy, all targets could be reached without traversing sulci, the insula, or lateral ventricles. Postoperative CT scans allowed visualization of the barium-impregnated tethers, and fusion of the scans with stereotactic MR images scan was used to verify the intended positions of the implants. Follow-up MRI at 3 and 12 months postimplantation showed no evidence of inflammation or device displacement. At 12 months, implants were successfully retrieved, and low but persistent NGF secretion was detected in half of the patients. With refinement, the ECB technology is positioned to become an important therapeutic platform in restorative neurosurgery and, in combination with other therapeutic factors, may be relevant for the treatment of a variety of neurological disorders. Clinical trial registration no.: NCT01163825.

Experimental Neurology, 2009

To date, a variety of pharmacological treatments exists for patients suffering epilepsy, but syst... more To date, a variety of pharmacological treatments exists for patients suffering epilepsy, but systemically administered drugs offer only symptomatic relief and often cause unwanted side effects. Moreover, available drugs are not effective in one third of the patients. Thus, more local and more effective treatment strategies need to be developed. Gene therapy-based expression of endogenous anti-epileptic agents represents a novel approach that could interfere with the disease process and result in stable and long-term suppression of seizures in epilepsy patients. We have reported earlier that direct in vivo viral vector-mediated overexpression of the glial cell line-derived neurotrophic factor (GDNF) in the rat hippocampus suppressed seizures in different animal models of epilepsy. Here we explored whether transplantation of encapsulated cells that release GDNF in the hippocampus could also exert a seizure-suppressant effect. Such ex vivo gene therapy approach represents a novel, more clinically safe approach, since the treatment could be terminated by retrieving the transplants from the brain. We demonstrate here that encapsulated cells, which are genetically modified to produce and release GDNF, can suppress recurrent generalized seizures when implanted into the hippocampus of kindled rats.

Aids, 1992

To map epitopes on gp120 defined by human antibodies and to examine the neutralizing activity of ... more To map epitopes on gp120 defined by human antibodies and to examine the neutralizing activity of these antibodies. Serum from HIV-1-antibody-positive individuals was used to screen a random fragment expression library representing gp120 from the HIVIIIB clone BH10. The library was based on the pUEX1 expression vector. Serum was tested for in vitro neutralizing activity using H9 cells and the HIVIIIB isolate. Four different epitopes defined by human antibodies were mapped on gp120. Two of these have not previously been reported and are located within amino acids (aa) 90-100 in the C1 region and aa 355-365 in the semi-conserved region between V3 and V4. The other two are located within aa 140-145 and aa 286-309. These epitopes are situated in regions that have been shown to demarcate human epitopes. Three serum samples with neutralization titres > or = 1024 were identified. None of the purified antibody fractions defining the mapped epitopes on gp120 had any neutralizing capacity against HIVIIIB. This study is the first demonstration of the applicability of random fragment expression libraries for the direct screening of human serum in order to map epitopes on gp120. Two new epitopes and two previously identified epitopes were mapped in this way. However, none of the linear epitopes was defined by antibody fractions neutralizing HIVIIIB, and it was not possible to map epitopes defined by neutralizing antibodies in the serum samples capable of neutralizing HIVIIIB infection of H9 cells. Thus, it appears that the neutralizing activity of serum in this study was not due to anti-gp120 antibodies defining linear epitopes.

Gene, 2002

The development of a set of synthetic mammalian promoters with different specific activities is d... more The development of a set of synthetic mammalian promoters with different specific activities is described. The library is based on a synthetic promoter, JeT, constructed as a 200 bp chimeric promoter built from fragments of the viral SV40 early promoter and the human bactin and ubiquitin C promoters. The JeT promoter was made by separating the included consensus boxes by the same distances in base pairs as found in the wild-type promoters, thus preserving transcription factor interaction. The resulting promoter was shown to drive reporter expression to high levels in enhanced green fluorescent protein and secreted alkaline phosphatase reporter assays. By replacing sequences separating the transcription factor binding sites with randomized sequences of the same length, sets of new promoters with different strengths, spanning a 10-fold range of transcriptional activity in cell culture, was obtained. The measured activity of each promoter in the library was highly specific and reproducible when tested in HiB5 and ARPE-19 cell culture. q

Molecular Therapy, 2010

Nerve growth factor (NGF) prevents cholinergic degeneration in Alzheimer's disease (AD) and impro... more Nerve growth factor (NGF) prevents cholinergic degeneration in Alzheimer's disease (AD) and improves memory in AD animal models. In humans, the safe delivery of therapeutic doses of NGF is challenging. For clinical use, we have therefore developed an encapsulated cell (EC) biodelivery device, capable of local delivery of NGF. The clinical device, named NsG0202, houses an NGF-secreting cell line (NGC-0295), which is derived from a human retinal pigment epithelial (RPE) cell line, stably genetically modified to secrete NGF. Bioactivity and correct processing of NGF was confirmed in vitro. NsG0202 devices were implanted in the basal forebrain of Göttingen minipigs and the function and retrievability were evaluated after 7 weeks, 6 and 12 months. All devices were implanted and retrieved without associated complications. They were physically intact and contained a high number of viable and NGF-producing NGC-0295 cells after explantation. Increased NGF levels were detected in tissue surrounding the devices. The implants were well tolerated as determined by histopathological brain tissue analysis, blood analysis, and general health status of the pigs. The NsG0202 device represents a promising approach for treating the cognitive decline in AD patients.

Molecular Biotechnology, 2005

During the past decade, lentiviral vectors based on the HIV-1 genome have been developed to becom... more During the past decade, lentiviral vectors based on the HIV-1 genome have been developed to become highly useful tools for efficient and stable delivery of transgenes to dividing and nondividing cells in a variety of experimental protocols. The vector system has been progressively and substantially improved, mainly to meet growing concerns over safety issues. However, the actual design and size of the lentiviral transfer vector often makes transgene cloning and DNA preparation a troublesome task. In this study, the pHR transfer vector used for lentivirus production in many laboratories was modified to contain a more versatile polylinker than the one present in the original pHR vector. In addition, the vector was significantly reduced in size from 12 to 7 kb, by replacing the original vector backbone with sequence from the multipurpose pUC18 vector. These modifications allowed for easier cloning and higher DNA yields without compromising the fundamental ability of this vector system to transduce cells in vitro and in vivo. Finally, the trimmed vector sequence was fully characterized by sequencing the vector in its entirety. In both cultured cells and directly into the rat striatum, transduction with this lentivirus, based on the modified pHsCXW vector, was as efficient and durable as with the pHR vector-based virus. In conclusion, the modified lentiviral transfer vector pHsCXW holds promise as a new valuable tool for the research community in the field of gene transfer.

Journal of Virological Methods, 1991

A novel competition ELBA for detection of antibodies against HIV-1 was developed. The assay is ba... more A novel competition ELBA for detection of antibodies against HIV-1 was developed. The assay is based on competition at the single epitope level and utilises a human monoclonal antibody and an E. co&produced fragment of the transmembrane glycoprotein gp41. The sensitivity of the assay was 100% in tests on 247 serum samples obtained from 219 individuals previously shown to be HIV-l antibody positive by both conventional indirect ELISA and the immunoblotting test. The patients represented various clinical and immunological stages of HIV-1 infection. Likewise, the specificity of the assay was 100% in tests on 105 serum samples from normal individuals previously tested negative by indirect ELISA. Further, among 105 serum samples selected due to consistent false positive reactions in the indirect ELISA only 2 samples (1.9%) demonstrated false positive reactions in the competition ELISA, i.e. 98.1% specificity. Finally, only 2 of 57 (3.5%) serum samples from HIV-2 infected individuals showed positive reactions in the assay, while 54 (94.7%) had absorbance values similar to the negative controls. These results demonstrate that human monoclonal antibodies may form the basis for highly sensitive and specific assays for detection of antibodies to HIV-1.

AIDS, 1992

To map epitopes on gp120 defined by human antibodies and to examine the neutralizing activity of ... more To map epitopes on gp120 defined by human antibodies and to examine the neutralizing activity of these antibodies. Serum from HIV-1-antibody-positive individuals was used to screen a random fragment expression library representing gp120 from the HIVIIIB clone BH10. The library was based on the pUEX1 expression vector. Serum was tested for in vitro neutralizing activity using H9 cells and the HIVIIIB isolate. Four different epitopes defined by human antibodies were mapped on gp120. Two of these have not previously been reported and are located within amino acids (aa) 90-100 in the C1 region and aa 355-365 in the semi-conserved region between V3 and V4. The other two are located within aa 140-145 and aa 286-309. These epitopes are situated in regions that have been shown to demarcate human epitopes. Three serum samples with neutralization titres > or = 1024 were identified. None of the purified antibody fractions defining the mapped epitopes on gp120 had any neutralizing capacity against HIVIIIB. This study is the first demonstration of the applicability of random fragment expression libraries for the direct screening of human serum in order to map epitopes on gp120. Two new epitopes and two previously identified epitopes were mapped in this way. However, none of the linear epitopes was defined by antibody fractions neutralizing HIVIIIB, and it was not possible to map epitopes defined by neutralizing antibodies in the serum samples capable of neutralizing HIVIIIB infection of H9 cells. Thus, it appears that the neutralizing activity of serum in this study was not due to anti-gp120 antibodies defining linear epitopes.

AIDS Research and Human Retroviruses, 1992

Three murine monoclonal antibodies (MAbs) F5-2, F5-4, and F5-16 defining three different epitopes... more Three murine monoclonal antibodies (MAbs) F5-2, F5-4, and F5-16 defining three different epitopes on the major core protein p24 of the human immunodeficiency virus type 1 (HIV-1) were epitope mapped using a random fragment expression library representing the p17- and p24-encoding part of the gag open reading frame. F5-2 defined an epitope within amino acids (aa) 14-23 at the N-terminus of p24, and F5-4 defined an epitope within aa 153-174 in the C-terminus of p24. F5-16 did not recognize any of the fusion proteins produced by the expression library indicating that this MAb defines a true conformational epitope on p24. Since the N-terminus of p24 has been reported to be immunosilent in humans, 356 HIV-1 antibody-positive serum samples were tested for reactivity against the region of p24 defined by F5-2. More than one third of the samples recognized this region indicating that it is immunoreactive and, further, the presence of antibodies against this region was associated with a reduced CD4 cell count.

The Open Tissue Engineering and Regenerative Medicine Journal, 2012

... Lone Fjord-Larsen*,1, Philip Kusk1, Malene Torp1, Jens Christian H. Sørensen2, Kaare Ettrup2,... more ... Lone Fjord-Larsen*,1, Philip Kusk1, Malene Torp1, Jens Christian H. Sørensen2, Kaare Ettrup2, Carsten R. Bjarkam2, Jens Tornøe1, Bengt ... Five female Göttingen minipigs (Ellegaard Göttingen Minipigs ApS, DK), weighing 7.8±0.2 kg at the start of the experiment, were ...

Journal of Bone and Mineral Metabolism, 2004

Experimental Cell Research, 2009

Neural stem cells (NSCs) are powerful research tools for the design and discovery of new approach... more Neural stem cells (NSCs) are powerful research tools for the design and discovery of new approaches to cell therapy in neurodegenerative diseases like Parkinson's disease. Several epigenetic and genetic strategies have been tested for long-term maintenance and expansion of these cells in vitro.

ABSTRACT The present invention relates to the field of therapeutic use of proteins, genes and cel... more ABSTRACT The present invention relates to the field of therapeutic use of proteins, genes and cells, in particular to the therapy based on secreted therapeutic proteins, NsG29 and NsG31. NsG29 and NsG31 are members of a newly identified family of growth factors with a specific cystein pattern and characterised by expression in the nevous system. The secreted growth factors have potential for the treatment of disorders of the nervous system. The invention also relates to bioactive NsG29 and NsG31 polypeptide fragments and the corresponding encoding DNA sequences

Experimental Neurology, 2005

Protein injection studies of the glial cell line derived neurotrophic factor (GDNF) family member... more Protein injection studies of the glial cell line derived neurotrophic factor (GDNF) family member Neurturin (NTN) have demonstrated neuroprotective effects on dopaminergic (DA) neurons, which are selectively lost during Parkinson's disease (PD). However, unlike GDNF, NTN has not previously been applied in PD models using an in vivo gene therapy approach. Difficulties with lentiviral gene delivery of wild type (wt) NTN led us to examine the role of the pre-pro-sequence, and to evaluate different NTN constructs in order to optimize gene therapy with NTN. Results from transfected cultured cells showed that wt NTN was poorly processed, and secreted as a pro-form. A similarly poor processing was found with a chimeric protein consisting of the pre-pro-part from GDNF and mature NTN. Moreover, we found that the biological activity of pro-NTN differs from mature NTN, as pro-NTN did not form a signaling complex with the tyrosine kinase receptor Ret and GFRa2 or GFRa1. Deletion of the pro-region resulted in significantly higher secretion of active NTN, which was further increased when substituting the wt NTN signal peptide with the immunoglobulin heavy-chain signal peptide (IgSP). The enhanced secretion of active mature NTN using the IgSP-NTN construct was reproduced in vivo in lentiviral-transduced rat striatal cells and, unlike wt NTN, enabled efficient neuroprotection of lesioned nigral DA neurons, similar to GDNF. An in vivo gene therapy approach with a modified NTN construct is therefore a possible treatment option for Parkinson's disease that should be further explored. D

Mol Ther, 2010

Nerve growth factor (NGF) prevents cholinergic degeneration in Alzheimer's disease (AD) and impro... more Nerve growth factor (NGF) prevents cholinergic degeneration in Alzheimer's disease (AD) and improves memory in AD animal models. In humans, the safe delivery of therapeutic doses of NGF is challenging. For clinical use, we have therefore developed an encapsulated cell (EC) biodelivery device, capable of local delivery of NGF. The clinical device, named NsG0202, houses an NGF-secreting cell line (NGC-0295), which is derived from a human retinal pigment epithelial (RPE) cell line, stably genetically modified to secrete NGF. Bioactivity and correct processing of NGF was confirmed in vitro. NsG0202 devices were implanted in the basal forebrain of Göttingen minipigs and the function and retrievability were evaluated after 7 weeks, 6 and 12 months. All devices were implanted and retrieved without associated complications. They were physically intact and contained a high number of viable and NGF-producing NGC-0295 cells after explantation. Increased NGF levels were detected in tissue surrounding the devices. The implants were well tolerated as determined by histopathological brain tissue analysis, blood analysis, and general health status of the pigs. The NsG0202 device represents a promising approach for treating the cognitive decline in AD patients.

Epilepsia, 2014

Litsa Nikitidou finished her PhD in Sweden and is currently a postdoctoral researcher in Hungary.

Molecular Therapy, 2010

Nerve growth factor (NGF) prevents cholinergic degeneration in Alzheimer's disease (AD) and impro... more Nerve growth factor (NGF) prevents cholinergic degeneration in Alzheimer's disease (AD) and improves memory in AD animal models. In humans, the safe delivery of therapeutic doses of NGF is challenging. For clinical use, we have therefore developed an encapsulated cell (EC) biodelivery device, capable of local delivery of NGF. The clinical device, named NsG0202, houses an NGF-secreting cell line (NGC-0295), which is derived from a human retinal pigment epithelial (RPE) cell line, stably genetically modified to secrete NGF. Bioactivity and correct processing of NGF was confirmed in vitro. NsG0202 devices were implanted in the basal forebrain of Göttingen minipigs and the function and retrievability were evaluated after 7 weeks, 6 and 12 months. All devices were implanted and retrieved without associated complications. They were physically intact and contained a high number of viable and NGF-producing NGC-0295 cells after explantation. Increased NGF levels were detected in tissue surrounding the devices. The implants were well tolerated as determined by histopathological brain tissue analysis, blood analysis, and general health status of the pigs. The NsG0202 device represents a promising approach for treating the cognitive decline in AD patients.

ABSTRACT Disclosed are NsG28, NsG30, NsG32 polypeptides, nucleic acids encoding NsG28, NsG30, NsG... more ABSTRACT Disclosed are NsG28, NsG30, NsG32 polypeptides, nucleic acids encoding NsG28, NsG30, NsG32 polypeptides, and antibodies that bind to NsG28, NsG30, NsG32 polypeptides as well as methods of making and using the same.

Journal of Neurosurgery, 2012

The authors describe the first clinical trial with encapsulated cell biodelivery (ECB) implants t... more The authors describe the first clinical trial with encapsulated cell biodelivery (ECB) implants that deliver nerve growth factor (NGF) to the cholinergic basal forebrain with the intention of halting the degeneration of cholinergic neurons and the associated cognitive decline in patients with Alzheimer disease (AD). The NsG0202 implant (NsGene A/S) consists of an NGF-producing, genetically engineered human cell line encapsulated behind a semipermeable hollow fiber membrane that allows the influx of nutrients and the efflux of NGF. The centimeter-long capsule is attached to an inert polymer tether that is used to guide the capsule to the target via stereotactic techniques and is anchored to the skull at the bur hole. Six patients with mild to moderate AD were included in this Phase Ib open-label safety study and were divided into 2 dose cohorts. The first cohort of 3 patients received single implants targeting the basal nucleus of Meynert (Ch4 region) bilaterally (2 implants per patient), and after a safety evaluation, a second cohort of 3 patients received bilateral implants (a total of 4 implants per patient) targeting both the Ch4 region and the vertical limb of the diagonal band of Broca (Ch2 region). Stereotactic implantation of the devices was successfully accomplished in all patients. Despite extensive brain atrophy, all targets could be reached without traversing sulci, the insula, or lateral ventricles. Postoperative CT scans allowed visualization of the barium-impregnated tethers, and fusion of the scans with stereotactic MR images scan was used to verify the intended positions of the implants. Follow-up MRI at 3 and 12 months postimplantation showed no evidence of inflammation or device displacement. At 12 months, implants were successfully retrieved, and low but persistent NGF secretion was detected in half of the patients. With refinement, the ECB technology is positioned to become an important therapeutic platform in restorative neurosurgery and, in combination with other therapeutic factors, may be relevant for the treatment of a variety of neurological disorders. Clinical trial registration no.: NCT01163825.

Experimental Neurology, 2009

To date, a variety of pharmacological treatments exists for patients suffering epilepsy, but syst... more To date, a variety of pharmacological treatments exists for patients suffering epilepsy, but systemically administered drugs offer only symptomatic relief and often cause unwanted side effects. Moreover, available drugs are not effective in one third of the patients. Thus, more local and more effective treatment strategies need to be developed. Gene therapy-based expression of endogenous anti-epileptic agents represents a novel approach that could interfere with the disease process and result in stable and long-term suppression of seizures in epilepsy patients. We have reported earlier that direct in vivo viral vector-mediated overexpression of the glial cell line-derived neurotrophic factor (GDNF) in the rat hippocampus suppressed seizures in different animal models of epilepsy. Here we explored whether transplantation of encapsulated cells that release GDNF in the hippocampus could also exert a seizure-suppressant effect. Such ex vivo gene therapy approach represents a novel, more clinically safe approach, since the treatment could be terminated by retrieving the transplants from the brain. We demonstrate here that encapsulated cells, which are genetically modified to produce and release GDNF, can suppress recurrent generalized seizures when implanted into the hippocampus of kindled rats.

Aids, 1992

To map epitopes on gp120 defined by human antibodies and to examine the neutralizing activity of ... more To map epitopes on gp120 defined by human antibodies and to examine the neutralizing activity of these antibodies. Serum from HIV-1-antibody-positive individuals was used to screen a random fragment expression library representing gp120 from the HIVIIIB clone BH10. The library was based on the pUEX1 expression vector. Serum was tested for in vitro neutralizing activity using H9 cells and the HIVIIIB isolate. Four different epitopes defined by human antibodies were mapped on gp120. Two of these have not previously been reported and are located within amino acids (aa) 90-100 in the C1 region and aa 355-365 in the semi-conserved region between V3 and V4. The other two are located within aa 140-145 and aa 286-309. These epitopes are situated in regions that have been shown to demarcate human epitopes. Three serum samples with neutralization titres > or = 1024 were identified. None of the purified antibody fractions defining the mapped epitopes on gp120 had any neutralizing capacity against HIVIIIB. This study is the first demonstration of the applicability of random fragment expression libraries for the direct screening of human serum in order to map epitopes on gp120. Two new epitopes and two previously identified epitopes were mapped in this way. However, none of the linear epitopes was defined by antibody fractions neutralizing HIVIIIB, and it was not possible to map epitopes defined by neutralizing antibodies in the serum samples capable of neutralizing HIVIIIB infection of H9 cells. Thus, it appears that the neutralizing activity of serum in this study was not due to anti-gp120 antibodies defining linear epitopes.

Gene, 2002

The development of a set of synthetic mammalian promoters with different specific activities is d... more The development of a set of synthetic mammalian promoters with different specific activities is described. The library is based on a synthetic promoter, JeT, constructed as a 200 bp chimeric promoter built from fragments of the viral SV40 early promoter and the human bactin and ubiquitin C promoters. The JeT promoter was made by separating the included consensus boxes by the same distances in base pairs as found in the wild-type promoters, thus preserving transcription factor interaction. The resulting promoter was shown to drive reporter expression to high levels in enhanced green fluorescent protein and secreted alkaline phosphatase reporter assays. By replacing sequences separating the transcription factor binding sites with randomized sequences of the same length, sets of new promoters with different strengths, spanning a 10-fold range of transcriptional activity in cell culture, was obtained. The measured activity of each promoter in the library was highly specific and reproducible when tested in HiB5 and ARPE-19 cell culture. q

Molecular Therapy, 2010

Nerve growth factor (NGF) prevents cholinergic degeneration in Alzheimer's disease (AD) and impro... more Nerve growth factor (NGF) prevents cholinergic degeneration in Alzheimer's disease (AD) and improves memory in AD animal models. In humans, the safe delivery of therapeutic doses of NGF is challenging. For clinical use, we have therefore developed an encapsulated cell (EC) biodelivery device, capable of local delivery of NGF. The clinical device, named NsG0202, houses an NGF-secreting cell line (NGC-0295), which is derived from a human retinal pigment epithelial (RPE) cell line, stably genetically modified to secrete NGF. Bioactivity and correct processing of NGF was confirmed in vitro. NsG0202 devices were implanted in the basal forebrain of Göttingen minipigs and the function and retrievability were evaluated after 7 weeks, 6 and 12 months. All devices were implanted and retrieved without associated complications. They were physically intact and contained a high number of viable and NGF-producing NGC-0295 cells after explantation. Increased NGF levels were detected in tissue surrounding the devices. The implants were well tolerated as determined by histopathological brain tissue analysis, blood analysis, and general health status of the pigs. The NsG0202 device represents a promising approach for treating the cognitive decline in AD patients.

Molecular Biotechnology, 2005

During the past decade, lentiviral vectors based on the HIV-1 genome have been developed to becom... more During the past decade, lentiviral vectors based on the HIV-1 genome have been developed to become highly useful tools for efficient and stable delivery of transgenes to dividing and nondividing cells in a variety of experimental protocols. The vector system has been progressively and substantially improved, mainly to meet growing concerns over safety issues. However, the actual design and size of the lentiviral transfer vector often makes transgene cloning and DNA preparation a troublesome task. In this study, the pHR transfer vector used for lentivirus production in many laboratories was modified to contain a more versatile polylinker than the one present in the original pHR vector. In addition, the vector was significantly reduced in size from 12 to 7 kb, by replacing the original vector backbone with sequence from the multipurpose pUC18 vector. These modifications allowed for easier cloning and higher DNA yields without compromising the fundamental ability of this vector system to transduce cells in vitro and in vivo. Finally, the trimmed vector sequence was fully characterized by sequencing the vector in its entirety. In both cultured cells and directly into the rat striatum, transduction with this lentivirus, based on the modified pHsCXW vector, was as efficient and durable as with the pHR vector-based virus. In conclusion, the modified lentiviral transfer vector pHsCXW holds promise as a new valuable tool for the research community in the field of gene transfer.