scholarly journals EXPRESSION OF A SECRETABLE, CELL-PENETRATING CDKL5 PROTEIN ENHANCES THE EFFICACY OF AAV VECTOR-MEDIATED GENE THERAPY FOR CDKL5 DEFICIENCY DISORDER.

2021 ◽  
Author(s):  
Giorgio Medici ◽  
Marianna Tassinari ◽  
Giuseppe Galvani ◽  
Stefano Bastianini ◽  
Laura Gennaccaro ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Neurodevelopmental Disorder ◽  
Monogenic Disease ◽  
Cyclin Dependent Kinase ◽  
Proof Of Concept ◽  
Wild Type ◽  
Infection Efficiency ◽  
Brain Infection ◽  
Cell Penetrating ◽  
Significant Efficacy

No therapy is currently available for CDKL5 (cyclin-dependent kinase-like 5) deficiency disorder (CDD), a severe neurodevelopmental disorder caused by mutations in the CDKL5 gene. Although delivery of a wild-type copy of the mutated gene to cells represents the most curative approach for a monogenic disease, proof-of-concept studies highlight significant efficacy caveats for brain gene therapy. Herein, we used a secretable TATk-CDKL5 protein to enhance the efficiency of a gene therapy for CDD. We found that, although AAVPHP.B_Igk-TATk-CDKL5 and AAVPHP.B_CDKL5 vectors had similar brain infection efficiency, the AAVPHP.B_Igk-TATk-CDKL5 vector led to a higher CDKL5 protein replacement due to secretion and transduction of the TATk-CDKL5 protein into the neighboring cells. Importantly, Cdkl5 KO mice treated with the AAVPHP.B_Igk-TATk-CDKL5 vector showed a behavioral and neuroanatomical improvement in comparison with vehicle-treated Cdkl5 KO mice or Cdkl5 KO mice treated with the AAVPHP.B_CDKL5 vector, indicating that a gene therapy based on a secretable recombinant TATk-CDKL5 protein is more effective at compensating Cdkl5-null brain defects than gene therapy based on the expression of the native CDKL5.

scholarly journals α-Galactosidase A Augmentation by Non-Viral Gene Therapy: Evaluation in Fabry Disease Mice

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 771
Author(s):  
Julen Rodríguez-Castejón ◽  
Ana Alarcia-Lacalle ◽  
Itziar Gómez-Aguado ◽  
Mónica Vicente-Pascual ◽  
María Ángeles Solinís Aspiazu ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Fabry Disease ◽  
Viral Vector ◽  
Viral Gene ◽  
Lysosomal Storage ◽  
Wild Type ◽  
Dose Administration ◽  
Viral Gene Therapy ◽  
Liver Transaminases ◽  
Low Levels

Fabry disease (FD) is a monogenic X-linked lysosomal storage disorder caused by a deficiency in the lysosomal enzyme α-Galactosidase A (α-Gal A). It is a good candidate to be treated with gene therapy, in which moderately low levels of enzyme activity should be sufficient for clinical efficacy. In the present work we have evaluated the efficacy of a non-viral vector based on solid lipid nanoparticles (SLN) to increase α-Gal A activity in an FD mouse model after intravenous administration. The SLN-based vector incremented α-Gal A activity to about 10%, 15%, 20% and 14% of the levels of the wild-type in liver, spleen, heart and kidney, respectively. In addition, the SLN-based vector significantly increased α-Gal A activity with respect to the naked pDNA used as a control in plasma, heart and kidney. The administration of a dose per week for three weeks was more effective than a single-dose administration. Administration of the SLN-based vector did not increase liver transaminases, indicative of a lack of toxicity. Additional studies are necessary to optimize the efficacy of the system; however, these results reinforce the potential of lipid-based nanocarriers to treat FD by gene therapy.

Arf6-mediated macropinocytosis enhanced suicide gene therapy of C16TAB-condensed Tat/pDNA nanoparticles in ovarian cancer

Nanoscale ◽  
2021 ◽  
Author(s):  
Zhe Sun ◽  
Jinhai Huang ◽  
Linjia Su ◽  
Jing Li ◽  
Fangzheng Qi ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Gene Therapy ◽  
Cancer Treatment ◽  
Plasmid Dna ◽  
Suicide Gene ◽  
Cell Penetrating Peptides ◽  
Therapeutic Gene ◽  
Hiv Tat ◽  
Efficient Delivery ◽  
Cell Penetrating

Using cell-penetrating peptides (CPPs), typically HIV-Tat, to deliver the therapeutic gene for cancer treatment has being hampered by low efficient delivery and complicated uptake route of plasmid DNA (pDNA). On...

scholarly journals Preclinical Development of Autologous Hematopoietic Stem Cell-Based Gene Therapy for Immune Deficiencies: A Journey from Mouse Cage to Bed Side

Pharmaceutics ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 549
Author(s):  
Laura Garcia-Perez ◽  
Anita Ordas ◽  
Kirsten Canté-Barrett ◽  
Pauline Meij ◽  
Karin Pike-Overzet ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Severe Combined Immunodeficiency ◽  
Good Manufacturing Practice ◽  
Proof Of Concept ◽  
Preclinical Development ◽  
Hematopoietic Stem ◽  
Immune Deficiencies ◽  
Suitable Vector

Recent clinical trials using patient’s own corrected hematopoietic stem cells (HSCs), such as for primary immunodeficiencies (Adenosine deaminase (ADA) deficiency, X-linked Severe Combined Immunodeficiency (SCID), X-linked chronic granulomatous disease (CGD), Wiskott–Aldrich Syndrome (WAS)), have yielded promising results in the clinic; endorsing gene therapy to become standard therapy for a number of diseases. However, the journey to achieve such a successful therapy is not easy, and several challenges have to be overcome. In this review, we will address several different challenges in the development of gene therapy for immune deficiencies using our own experience with Recombinase-activating gene 1 (RAG1) SCID as an example. We will discuss product development (targeting of the therapeutic cells and choice of a suitable vector and delivery method), the proof-of-concept (in vitro and in vivo efficacy, toxicology, and safety), and the final release steps to the clinic (scaling up, good manufacturing practice (GMP) procedures/protocols and regulatory hurdles).

Thymidine kinase-mediated killing of rat brain tumors

1993 ◽  
Vol 79 (5) ◽  
pp. 729-735 ◽  
Author(s):  
David Barba ◽  
Joseph Hardin ◽  
Jasodhara Ray ◽  
Fred H. Gage
Keyword(s):  
Gene Therapy ◽  
Brain Tumors ◽  
Tumor Cells ◽  
Wild Type ◽  
Cns Disorders ◽  
Content Type ◽  
Potential Applications ◽  
Ganciclovir Treatment ◽  
The Brain ◽  
Fine Print

✓ Gene therapy has many potential applications in central nervous system (CNS) disorders, including the selective killing of tumor cells in the brain. A rat brain tumor model was used to test the herpes simplex virus (HSV)-thymidine kinase (TK) gene for its ability to selectively kill C6 and 9L tumor cells in the brain following systemic administration of the nucleoside analog ganciclovir. The HSV-TK gene was introduced in vitro into tumor cells (C6-TK and 9L-TK), then these modified tumor cells were evaluated for their sensitivity to cell killing by ganciclovir. In a dose-response assay, both C6-TK and 9L-TK cells were 100 times more sensitive to killing by ganciclovir (median lethal dose: C6-TK, 0.1 µg ganciclovir/ml; C6, 5.0 µg ganciclovir/ml) than unmodified wild-type tumor cells or cultured fibroblasts. In vivo studies confirmed the ability of intraperitoneal ganciclovir administration to kill established brain tumors in rats as quantified by both stereological assessment of brain tumor volumes and studies of animal survival over 90 days. Rats with brain tumors established by intracerebral injection of wild-type or HSV-TK modified tumor cells or by a combination of wild-type and HSV-TK-modified cells were studied with and without ganciclovir treatments. Stereological methods determined that ganciclovir treatment eliminated tumors composed of HSV-TK-modified cells while control tumors grew as expected (p < 0.001). In survival studies, all 10 rats with 9L-TK tumors treated with ganciclovir survived 90 days while all untreated rats died within 25 days. Curiously, tumors composed of combinations of 9L and 9L-TK cells could be eliminated by ganciclovir treatments even when only one-half of the tumor cells carried the HSV-TK gene. While not completely understood, this additional tumor cell killing appears to be both tumor selective and local in nature. It is concluded that HSV-TK gene therapy with ganciclovir treatment does selectively kill tumor cells in the brain and has many potential applications in CNS disorders, including the treatment of cancer.

scholarly journals Adeno-associated virus Rep proteins antagonize phosphatase PP1 to counteract KAP1 repression of the latent viral genome

2018 ◽  
Vol 115 (15) ◽  
pp. E3529-E3538 ◽  
Author(s):  
Sarah Smith-Moore ◽  
Stuart J. D. Neil ◽  
Cornel Fraefel ◽  
R. Michael Linden ◽  
Mathieu Bollen ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Helper Virus ◽  
Protein Phosphatase 1 ◽  
Wild Type ◽  
Fha Domain ◽  
Adeno Associated Virus ◽  
Histone 3 ◽  
Rep Proteins ◽  
Cellular Factors

Adeno-associated virus (AAV) is a small human Dependovirus whose low immunogenicity and capacity for long-term persistence have led to its widespread use as vector for gene therapy. Despite great recent successes in AAV-based gene therapy, further improvements in vector technology may be hindered by an inadequate understanding of various aspects of basic AAV biology. AAV is unique in that its replication is largely dependent on a helper virus and cellular factors. In the absence of helper virus coinfection, wild-type AAV establishes latency through mechanisms that are not yet fully understood. Challenging the currently held model for AAV latency, we show here that the corepressor Krüppel-associated box domain-associated protein 1 (KAP1) binds the latent AAV2 genome at the rep ORF, leading to trimethylation of AAV2-associated histone 3 lysine 9 and that the inactivation of KAP1 repression is necessary for AAV2 reactivation and replication. We identify a viral mechanism for the counteraction of KAP1 in which interference with the KAP1 phosphatase protein phosphatase 1 (PP1) by the AAV2 Rep proteins mediates enhanced phosphorylation of KAP1-S824 and thus relief from KAP1 repression. Furthermore, we show that this phenomenon involves recruitment of the NIPP1 (nuclear inhibitor of PP1)–PP1α holoenzyme to KAP1 in a manner dependent upon the NIPP1 FHA domain, identifying NIPP1 as an interaction partner for KAP1 and shedding light on the mechanism through which PP1 regulates cellular KAP1 activity.

scholarly journals Transferrin-Conjugated SNALPs Encapsulating 2′-O-Methylated miR-34a for the Treatment of Multiple Myeloma

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Immacolata Scognamiglio ◽  
Maria Teresa Di Martino ◽  
Virginia Campani ◽  
Antonella Virgilio ◽  
Aldo Galeone ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Zeta Potential ◽  
Lipid Vesicles ◽  
Significant Inhibition ◽  
The Other ◽  
Transferrin Receptors ◽  
Proof Of Concept ◽  
Wild Type ◽  
Chemical Conjugation ◽  
Mean Size

Stable nucleic acid lipid vesicles (SNALPs) encapsulating miR-34a to treat multiple myeloma (MM) were developed. Wild type or completely 2′-O-methylated (OMet) MiR-34a was used in this study. Moreover, SNALPs were conjugated with transferrin (Tf) in order to target MM cells overexpressing transferrin receptors (TfRs). The type of miR-34a chemical backbone did not significantly affect the characteristics of SNALPs in terms of mean size, polydispersity index, and zeta potential, while the encapsulation of an OMet miR-34a resulted in a significant increase of miRNA encapsulation into the SNALPs. On the other hand, the chemical conjugation of SNALPs with Tf resulted in a significant decrease of the zeta potential, while size characteristics and miR-34a encapsulation into SNALPs were not significantly affected. In an experimental model of MM, all the animals treated with SNALPs encapsulating miR-34a showed a significant inhibition of the tumor growth. However, the use of SNALPs conjugated with Tf and encapsulating OMet miR-34a resulted in the highest increase of mice survival. These results may represent the proof of concept for the use of SNALPs encapsulating miR-34a for the treatment of MM.

scholarly journals Hemophilia gene therapy: ushering in a new treatment paradigm?

Hematology ◽  
2021 ◽  
Vol 2021 (1) ◽  
pp. 226-233
Author(s):  
Lindsey A. George
Keyword(s):  
Clinical Trial ◽  
Gene Therapy ◽  
Clinical Trials ◽  
Hemophilia A ◽  
Full Potential ◽  
Proof Of Concept ◽  
Aav Vector ◽  
Treatment Paradigm ◽  
Clinical Trial Enrollment ◽  
New Treatment

Abstract After 3 decades of clinical trials, repeated proof-of-concept success has now been demonstrated in hemophilia A and B gene therapy. Current clinical hemophilia gene therapy efforts are largely focused on the use of systemically administered recombinant adeno-associated viral (rAAV) vectors for F8 or F9 gene addition. With multiple ongoing trials, including licensing studies in hemophilia A and B, many are cautiously optimistic that the first AAV vectors will obtain regulatory approval within approximately 1 year. While supported optimism suggests that the goal of gene therapy to alter the paradigm of hemophilia care may soon be realized, a number of outstanding questions have emerged from clinical trial that are in need of answers to harness the full potential of gene therapy for hemophilia patients. This article reviews the use of AAV vector gene addition approaches for hemophilia A and B, focusing specifically on information to review in the process of obtaining informed consent for hemophilia patients prior to clinical trial enrollment or administering a licensed AAV vector.

scholarly journals Daily oscillation of the excitation/inhibition ratio is disrupted in two mouse models of autism

2021 ◽  
Author(s):  
Michelle Bridi ◽  
Nancy Luo ◽  
Grace Kim ◽  
Caroline O'Ferrall ◽  
Ruchit Oatel ◽  
...  
Keyword(s):  
Mouse Models ◽  
Sensory Processing ◽  
Neurodevelopmental Disorder ◽  
Autism Spectrum ◽  
Behavioral State ◽  
Wild Type ◽  
Inhibitory Synaptic Transmission ◽  
Sleep Timing ◽  
Per Se ◽  
Daily Oscillation

Autism spectrum disorder (ASD) is a prevalent neurodevelopmental disorder involving sensory processing abnormalities. Alterations to the balance between excitation and inhibition (E/I ratio) are postulated to underlie behavioral phenotypes in ASD patients and mouse models. However, in primary visual cortex (V1) of wild type mice, the E/I ratio is not a fixed value, but rather oscillates across the 24h day. Therefore, we hypothesized that the E/I oscillation, rather than the overall E/I ratio, may be disrupted in ASD mouse models. To this end, we measured the E/I ratio in Fmr1 KO and BTBR mice, models of syndromic and idiopathic ASD, respectively. We found that the E/I ratio is dysregulated in both models, but in different ways: the oscillation is flattened in Fmr1 KO and phase-shifted in BTBR mice. These phenotypes cannot be explained by altered sleep timing, which was largely normal in both lines. Furthermore, we found that E/I dysregulation occurs due to alterations in both excitatory and inhibitory synaptic transmission in both models. These findings provide a crucial perspective on the E/I ratio in ASD, suggesting that ASD phenotypes may be produced by a mismatch of E/I to the appropriate behavioral state, rather than alterations to overall E/I levels per se.

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