scholarly journals The development and characterization of an scFv-Fc fusion based gene therapy to reduce the psychostimulant effects of methamphetamine abuse

2019 ◽  
Author(s):  
Charles E. Hay ◽  
Laura E. Ewing ◽  
Michael D. Hambuchen ◽  
Shannon M. Zintner ◽  
Juliana C. Small ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Ex Vivo ◽  
Fusion Construct ◽  
Biodistribution Studies ◽  
Time Period ◽  
Alternative Approach ◽  
The Us ◽  
Abused Drugs ◽  
High Doses

AbstractMethamphetamine (METH) continues to be amongst the most addictive and abused drugs in the US. Unfortunately, there are currently no FDA approved pharmacological treatments for METH substance abuse disorder. As an alternative approach, we have previously explored the use of Adeno-associated viral (AAV) mediated gene transfer of an anti-METH monoclonal antibody. Here, we advance our approach by generating a novel anti-METH scFv-Fc fusion construct (7F9-Fc), packaged into AAV serotype 8 vector (called AAV-scFv-Fc), and tested in vivo and ex vivo. A range of doses (1 × 1010. 1 × 1011, and 1 × 1012 vector copies(vc)/mouse) were administered to mice, which exhibited a dose-dependent expression of 7F9-Fc in serum with peak circulating concentrations of 48, 1785, and 3,831 μg/ml. The dose of 1 × 1012 vc/mouse was further tested in METH locomotor and biodistribution studies to determine the efficacy of the antibody protection. Expressed 7F9-Fc exhibited high affinity binding, 17 nM, to METH. Between days 21 and 35 after vector administration, the 7F9-Fc gene therapy significantly reduced the effects of METH in locomotor assays following administration of moderate and high doses of subcutaneous METH, 3.1 and 9.4 mg/kg respectively. On day 116 post-AAV administration, mice expressing 7F9-Fc sequestered over 2.5 times more METH into the serum than vehicle mice, and METH concentrations in the brain were reduced by 1.2 times compared to vehicle mice. Taken together, these data suggest that a AAV-delivered anti-METH Fc fusion antibody could be a design for persistently reducing concentrations of METH in the CNS.Significance StatementIn this manuscript, we describe the use of a novel anti-METH scFv-Fc fusion protein delivered in mice using gene therapy. The results suggest that the gene therapy delivery system can lead to the production of enough antibody to mitigate METH’s psychostimulant effects in mice over an extended time period.

In Vivo Effect of Suloctidil as an Antiplatelet Agent

1979 ◽  
Vol 41 (03) ◽  
pp. 465-474 ◽  
Author(s):  
Marcia R Stelzer ◽  
Thomas S Burns ◽  
Robert N Saunders
Keyword(s):  
Ex Vivo ◽  
Antiplatelet Agent ◽  
Ratio Method ◽  
Platelet Aggregate ◽  
Permanent Effect ◽  
Platelet Aggregates ◽  
5 Ht Uptake ◽  
High Doses

SummaryThe relationship between the effects of suloctidil in vivo as an antiplatelet agent and in vitro as a modifier of platelet serotonin (5-HT) parameters was investigated. Suloctidil was found to be effective in reducing platelet aggregates formation in the retired breeder rat as determined using the platelet aggregate ratio method (PAR) with an ED50 of 16.1 mg/kg 24 hours post administration. In contrast to the hypothesis that 5-HT depletion is involved in the anti-aggregatory mechanism of suloctidil, no correlation was found between platelet 5- HT content and this antiplatelet activity. Reduction of platelet 5-HT content required multiple injections of high doses (100 mg/kg/day) of suloctidil. Suloctidil administration for 8 days at 100 mg/kg/day, which lowered platelet 5-HT content by 50%, resulted in no permanent effect on ex vivo platelet 5-HT uptake or thrombin-induced release, nor alteration in the plasma 5-HT level. However, these platelets exhibited a short-lived, significant increase in percent leakage of 5-HT after 30 minutes of incubation. Therefore, suloctidil treatment at high doses may with time result in platelet 5-HT depletion, however this effect is probably not related to the primary anti-aggregatory activity of the drug.

scholarly journals Effect of ethanol and cocaine on [11C]MPC-6827 uptake in SH-SY5Y cells

2021 ◽  
Author(s):  
Naresh Damuka ◽  
Miranda Orr ◽  
Paul W. Czoty ◽  
Jeffrey L. Weiner ◽  
Thomas J. Martin ◽  
...  
Keyword(s):  
Mechanism Of Action ◽  
Ex Vivo ◽  
Neuroblastoma Cells ◽  
Proper Function ◽  
Brain Functions ◽  
Biodistribution Studies ◽  
Positron Emission ◽  
Vitro Binding

AbstractMicrotubules (MTs) are structural units in the cytoskeleton. In brain cells they are responsible for axonal transport, information processing, and signaling mechanisms. Proper function of these processes is critical for healthy brain functions. Alcohol and substance use disorders (AUD/SUDs) affects the function and organization of MTs in the brain, making them a potential neuroimaging marker to study the resulting impairment of overall neurobehavioral and cognitive processes. Our lab reported the first brain-penetrant MT-tracking Positron Emission Tomography (PET) ligand [11C]MPC-6827 and demonstrated its in vivo utility in rodents and non-human primates. To further explore the in vivo imaging potential of [11C]MPC-6827, we need to investigate its mechanism of action. Here, we report preliminary in vitro binding results in SH-SY5Y neuroblastoma cells exposed to ethanol (EtOH) or cocaine in combination with multiple agents that alter MT stability. EtOH and cocaine treatments increased MT stability and decreased free tubulin monomers. Our initial cell-binding assay demonstrated that [11C]MPC-6827 may have high affinity to free/unbound tubulin units. Consistent with this mechanism of action, we observed lower [11C]MPC-6827 uptake in SH-SY5Y cells after EtOH and cocaine treatments (e.g., fewer free tubulin units). We are currently performing in vivo PET imaging and ex vivo biodistribution studies in rodent and nonhuman primate models of AUD and SUDs and Alzheimer's disease.

scholarly journals Growth Factor Therapy for Parkinson’s Disease: Alternative Delivery Systems

2021 ◽  
pp. 1-7
Author(s):  
Sarah Jarrin ◽  
Abrar Hakami ◽  
Ben Newland ◽  
Eilís Dowd
Keyword(s):  
Parkinson’S Disease ◽  
Gene Therapy ◽  
Parkinson's Disease ◽  
Growth Factors ◽  
Growth Factor ◽  
Ex Vivo ◽  
Brain Regions ◽  
Delivery Systems ◽  
Alternative Delivery

Despite decades of research and billions in global investment, there remains no preventative or curative treatment for any neurodegenerative condition, including Parkinson’s disease (PD). Arguably, the most promising approach for neuroprotection and neurorestoration in PD is using growth factors which can promote the growth and survival of degenerating neurons. However, although neurotrophin therapy may seem like the ideal approach for neurodegenerative disease, the use of growth factors as drugs presents major challenges because of their protein structure which creates serious hurdles related to accessing the brain and specific targeting of affected brain regions. To address these challenges, several different delivery systems have been developed, and two major approaches—direct infusion of the growth factor protein into the target brain region and in vivo gene therapy—have progressed to clinical trials in patients with PD. In addition to these clinically evaluated approaches, a range of other delivery methods are in various degrees of development, each with their own unique potential. This review will give a short overview of some of these alternative delivery systems, with a focus on ex vivo gene therapy and biomaterial-aided protein and gene delivery, and will provide some perspectives on their potential for clinical development and translation.

scholarly journals Development of alternative gene transfer techniques for ex vivo and in vivo gene therapy in a canine model

2021 ◽  
Vol 18 ◽  
pp. 347-354
Author(s):  
Masashi Noda ◽  
Kohei Tatsumi ◽  
Hideto Matsui ◽  
Yasunori Matsunari ◽  
Takeshi Sato ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Canine Model ◽  
Gene Transfer Techniques

scholarly journals Influence of Molecular Design on the Targeting Properties of ABD-Fused Mono- and Bi-Valent Anti-HER3 Affibody Therapeutic Constructs

Cells ◽  
2018 ◽  
Vol 7 (10) ◽  
pp. 164 ◽  
Author(s):  
Mohamed Altai ◽  
Charles Leitao ◽  
Sara Rinne ◽  
Anzhelika Vorobyeva ◽  
Christina Atterby ◽  
...  
Keyword(s):  
Half Life ◽  
Molecular Design ◽  
Growth Factor Receptor ◽  
Fusion Construct ◽  
Affibody Molecules ◽  
Binding Domains ◽  
Biodistribution Studies ◽  
C Terminus ◽  
Type 3

Overexpression of human epidermal growth factor receptor type 3 (HER3) is associated with tumour cell resistance to HER-targeted therapies. Monoclonal antibodies (mAbs) targeting HER3 are currently being investigated for treatment of various types of cancers. Cumulative evidence suggests that affibody molecules may be appropriate alternatives to mAbs. We previously reported a fusion construct (3A3) containing two HER3-targeting affibody molecules flanking an engineered albumin-binding domain (ABD035) included for the extension of half-life in circulation. The 3A3 fusion protein (19.7 kDa) was shown to delay tumour growth in mice bearing HER3-expressing xenografts and was equipotent to the mAb seribantumab. Here, we have designed and explored a series of novel formats of anti-HER3 affibody molecules fused to the ABD in different orientations. All constructs inhibited heregulin-induced phosphorylation in HER3-expressing BxPC-3 and DU-145 cell lines. Biodistribution studies demonstrated extended the half-life of all ABD-fused constructs, although at different levels. The capacity of our ABD-fused proteins to accumulate in HER3-expressing tumours was demonstrated in nude mice bearing BxPC-3 xenografts. Formats where the ABD was located on the C-terminus of affibody binding domains (3A, 33A, and 3A3) provided the best tumour targeting properties in vivo. Further development of these promising candidates for treatment of HER3-overexpressing tumours is therefore justified.

scholarly journals Visualisation of interstitial lung disease by molecular imaging of integrin αvβ3 and somatostatin receptor 2

2018 ◽  
Vol 78 (2) ◽  
pp. 218-227 ◽  
Author(s):  
Janine Schniering ◽  
Martina Benešová ◽  
Matthias Brunner ◽  
Stephanie Haller ◽  
Susan Cohrs ◽  
...  
Keyword(s):  
Interstitial Lung Disease ◽  
Lung Disease ◽  
Single Photon ◽  
Ex Vivo ◽  
Somatostatin Receptor ◽  
Photon Emission ◽  
Nuclear Imaging ◽  
Integrin Αvβ3 ◽  
Biodistribution Studies

ObjectiveTo evaluate integrin αvβ3 (alpha-v-beta-3)-targeted and somatostatin receptor 2 (SSTR2)-targeted nuclear imaging for the visualisation of interstitial lung disease (ILD).MethodsThe pulmonary expression of integrin αvβ3 and SSTR2 was analysed in patients with different forms of ILD as well as in bleomycin (BLM)-treated mice and respective controls using immunohistochemistry. Single photon emission CT/CT (SPECT/CT) was performed on days 3, 7 and 14 after BLM instillation using the integrin αvβ3-targeting 177Lu-DOTA-RGD and the SSTR2-targeting 177Lu-DOTA-NOC radiotracer. The specific pulmonary accumulation of the radiotracers over time was assessed by in vivo and ex vivo SPECT/CT scans and by biodistribution studies.ResultsExpression of integrin αvβ3 and SSTR2 was substantially increased in human ILD regardless of the subtype. Similarly, in lungs of BLM-challenged mice, but not of controls, both imaging targets were stage-specifically overexpressed. While integrin αvβ3 was most abundantly upregulated on day 7, the inflammatory stage of BLM-induced lung fibrosis, SSTR2 expression peaked on day 14, the established fibrotic stage. In agreement with the findings on tissue level, targeted nuclear imaging using SPECT/CT specifically detected both imaging targets ex vivo and in vivo, and thus visualised different stages of experimental ILD.ConclusionOur preclinical proof-of-concept study suggests that specific visualisation of molecular processes in ILD by targeted nuclear imaging is feasible. If transferred into clinics, where imaging is considered an integral part of patients’ management, the additional information derived from specific imaging tools could represent a first step towards precision medicine in ILD.

Gene therapy for critical limb ischemia: Per aspera ad astra

2021 ◽  
Vol 21 ◽  
Author(s):  
Vyacheslav Z. Tarantul ◽  
Alexander V. Gavrilenko
Keyword(s):  
Gene Therapy ◽  
Critical Limb Ischemia ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Single Gene ◽  
Public Health Problem ◽  
Limb Ischemia ◽  
Therapeutic Angiogenesis ◽  
Effective Treatment Option

: Peripheral artery diseases remain a serious public health problem. Although there are many traditional methods for their treatment using conservative therapeutic techniques and surgery, gene therapy is an alternative and potentially more effective treatment option especially for “no option” patients. This review treats the results of many years of research and application of gene therapy as an example of treatment of patients with critical limb ischemia. Data on successful and unsuccessful attempts to use this technology for treating this disease are presented. Trends in changing the paradigm of approaches to therapeutic angiogenesis are noted: from viral vectors to non-viral vectors, from gene transfer to the whole organism to targeted transfer to cells and tissues, from single gene use to combination of genes; from DNA therapy to RNA therapy, from in vivo therapy to ex vivo therapy.

scholarly journals Delivery of human factor IX in mice by encapsulated recombinant myoblasts: a novel approach towards allogeneic gene therapy of hemophilia B

Blood ◽  
1996 ◽  
Vol 87 (12) ◽  
pp. 5095-5103 ◽  
Author(s):  
G Hortelano ◽  
A Al-Hendy ◽  
FA Ofosu ◽  
PL Chang
Keyword(s):  
Gene Therapy ◽  
Human Factor ◽  
Ex Vivo ◽  
Cost Effective ◽  
Factor Ix ◽  
Hemophilia B ◽  
Therapy Strategy ◽  
Human Factor Ix

A potentially cost-effective strategy for gene therapy of hemophilia B is to create universal factor IX-secreting cell lines suitable for implantation into different patients. To avoid graft rejection, the implanted cells are enclosed in alginate-polylysine-alginate microcapsules that are permeable to factor IX diffusion, but impermeable to the hosts' immune mediators. This nonautologous approach was assessed by implanting encapsulated mouse myoblasts secreting human factor IX into allogeneic mice. Human factor IX was detected in the mouse plasma for up to 14 days maximally at approximately 4 ng/mL. Antibodies to human factor IX were detected after 3 weeks at escalating levels, which were sustained throughout the entire experiment (213 days). The antibodies accelerated the clearance of human factor IX from the circulation of the implanted mice and inhibited the detection of human factor IX in the mice plasma in vitro. The encapsulated myoblasts retrieved periodically from the implanted mice up to 213 days postimplantation were viable and continued to secrete human factor IX ex vivo at undiminished rates, hence suggesting continued factor IX gene expression in vivo. Thus, this allogeneic gene therapy strategy represents a potentially feasible alternative to autologous approaches for the treatment of hemophilia B.

scholarly journals Adenosine A2AReceptors in Substance Use Disorders: A Focus on Cocaine

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1372 ◽  
Author(s):  
Karolina Wydra ◽  
Dawid Gawliński ◽  
Kinga Gawlińska ◽  
Małgorzata Frankowska ◽  
Dasiel O. Borroto-Escuela ◽  
...  
Keyword(s):  
Substance Use ◽  
Substance Use Disorders ◽  
Current Knowledge ◽  
Ex Vivo ◽  
D2 Receptors ◽  
Future Research ◽  
Receptor Complexes ◽  
Abused Drugs ◽  
The Brain

Several psychoactive drugs can evoke substance use disorders (SUD) in humans and animals, and these include psychostimulants, opioids, cannabinoids (CB), nicotine, and alcohol. The etiology, mechanistic processes, and the therapeutic options to deal with SUD are not well understood. The common feature of all abused drugs is that they increase dopamine (DA) neurotransmission within the mesocorticolimbic circuitry of the brain followed by the activation of DA receptors. D2 receptors were proposed as important molecular targets for SUD. The findings showed that D2 receptors formed heteromeric complexes with other GPCRs, which forced the addiction research area in new directions. In this review, we updated the view on the brain D2 receptor complexes with adenosine (A)2A receptors (A2AR) and discussed the role of A2AR in different aspects of addiction phenotypes in laboratory animal procedures that permit the highly complex syndrome of human drug addiction. We presented the current knowledge on the neurochemical in vivo and ex vivo mechanisms related to cocaine use disorder (CUD) and discussed future research directions for A2AR heteromeric complexes in SUD.

Clonal Analyses of Retroviral Vector Integrations in ADA-SCID Patients Treated with Stem Cell Gene Therapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3249-3249
Author(s):  
Barbara Cassani ◽  
Grazia Andolfi ◽  
Massimiliano Mirolo ◽  
Luca Biasco ◽  
Alessandra Recchia ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Human Genome ◽  
Ex Vivo ◽  
Cd34 Cells

Abstract Gene transfer into hematopoietic stem/progenitor cells (HSC) by gammaretroviral vectors is an effective treatment for patients affected by severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA)-deficiency. Recent studied have indicated that gammaretroviral vectors integrate in a non-random fashion in their host genome, but there is still limited information on the distribution of retroviral insertion sites (RIS) in human long-term reconstituting HSC following therapeutic gene transfer. We performed a genome-wide analysis of RIS in transduced bone marrow-derived CD34+ cells before transplantation (in vitro) and in hematopoietic cell subsets (ex vivo) from five ADA-SCID patients treated with gene therapy combined to low-dose busulfan. Vector-genome junctions were cloned by inverse or linker-mediated PCR, sequenced, mapped onto the human genome, and compared to a library of randomly cloned human genome fragments or to the expected distribution for the NCBI annotation. Both in vitro (n=212) and ex vivo (n=496) RIS showed a non-random distribution, with strong preference for a 5-kb window around transcription start sites (23.6% and 28.8%, respectively) and for gene-dense regions. Integrations occurring inside the transcribed portion of a RefSeq genes were more represented in vitro than ex vivo (50.9 vs 41.3%), while RIS <30kb upstream from the start site were more frequent in the ex vivo sample (25.6% vs 19.4%). Among recurrently hit loci (n=50), LMO2 was the most represented, with one integration cloned from pre-infusion CD34+ cells and five from post-gene therapy samples (2 in granulocytes, 3 in T cells). Clone-specific Q-PCR showed no in vivo expansion of LMO2-carrying clones while LMO2 gene overexpression at the bulk level was excluded by RT-PCR. Gene expression profiling revealed a preference for integration into genes transcriptionally active in CD34+ cells at the time of transduction as well as genes expressed in T cells. Functional clustering analysis of genes hit by retroviral vectors in pre- and post-transplant cells showed no in vivo skewing towards genes controlling self-renewal or survival of HSC (i.e. cell cycle, transcription, signal transduction). Clonal analysis of long-term repopulating cells (>=6 months) revealed a high number of distinct RIS (range 42–121) in the T-cell compartment, in agreement with the complexity of the T-cell repertoire, while fewer RIS were retrieved from granulocytes. The presence of shared integrants among multiple lineages confirmed that the gene transfer protocol was adequate to allow stable engraftment of multipotent HSC. Taken together, our data show that transplantation of ADA-transduced HSC does not result in skewing or expansion of malignant clones in vivo, despite the occurrence of insertions near potentially oncogenic genomic sites. These results, combined to the relatively long-term follow-up of patients, indicate that retroviral-mediated gene transfer for ADA-SCID has a favorable safety profile.

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