scholarly journals Considerations for Preclinical Safety Assessment of Adeno-Associated Virus Gene Therapy Products

2018 ◽  
Vol 46 (8) ◽  
pp. 1020-1027 ◽  
Author(s):  
Basel T. Assaf ◽  
Laurence O. Whiteley
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Metabolic Diseases ◽  
Adeno Associated Virus ◽  
Monogenic Disorders ◽  
Iatrogenic Complications ◽  
Health And Disease ◽  
Molecular Bases ◽  
Preclinical Safety

Progress in understanding the molecular bases of human health and disease in recent decades has flourished making it possible for the field of gene therapy (GT) to offer new possibilities for treating, and even curing, a plethora of medical conditions such as monogenic disorders and metabolic diseases. GT is a therapeutic intervention to genetically alter or modify living cells by means of gene delivery achieved using either viral vectors or nonviral vectors, with adeno-associated virus (AAV) vectors constituting market-share majority. Although GT is conceptually attractive, adverse and even fatal iatrogenic complications have marred the initial enthusiasm of clinical successes. The properties of investigational AAV-based GT may pose safety concerns unique from those of small molecule drugs and other macromolecular biologics, such as ectopic or unregulated expression of the transgene, long-term persistence, and off-target distribution. Herein, we discuss considerations in the design of a comprehensive preclinical safety program for AAV-based GT prior to administration in humans.

Adeno-Associated Viral Vectors for Gene Transfer and Gene Therapy

1999 ◽  
Vol 380 (6) ◽  
Author(s):  
H. Büeler
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Gene Delivery ◽  
Viral Vectors ◽  
Delivery Systems ◽  
Adeno Associated Virus ◽  
Viral Genes ◽  
Virus Recombinant

AbstractAdeno-associated virus (AAV) is a defective, non-pathogenic human parvovirus that depends for growth on coinfection with a helper adenovirus or herpes virus. Recombinant adeno-associated viruses (rAAVs) have attracted considerable interest as vectors for gene therapy. In contrast to other gene delivery systems, rAAVs lack all viral genes and show long-term gene expression

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 Complete Correction of Brain and Spinal Cord Pathology in Metachromatic Leukodystrophy Mice

2021 ◽  
Vol 14 ◽  
Author(s):  
Emilie Audouard ◽  
Valentin Oger ◽  
Béatrix Meha ◽  
Nathalie Cartier ◽  
Caroline Sevin ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Gene Therapy ◽  
Metachromatic Leukodystrophy ◽  
Early Death ◽  
Lysosomal Storage ◽  
Adeno Associated Virus ◽  
Virus Serotype ◽  
Brain And Spinal Cord ◽  
Sulfatide Accumulation

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder characterized by accumulation of sulfatides in both glial cells and neurons. MLD results from an inherited deficiency of arylsulfatase A (ARSA) and myelin degeneration in the central and peripheral nervous systems. Currently, no effective treatment is available for the most frequent late infantile (LI) form of MLD after symptom onset. The LI form results in rapid neurological degradation and early death. ARSA enzyme must be rapidly and efficiently delivered to brain and spinal cord oligodendrocytes of patients with LI MLD in order to potentially stop the progression of the disease. We previously showed that brain gene therapy with adeno-associated virus serotype rh10 (AAVrh10) driving the expression of human ARSA cDNA alleviated most long-term disease manifestations in MLD mice but was not sufficient in MLD patient to improve disease progression. Herein, we evaluated the short-term effects of intravenous AAVPHP.eB delivery driving the expression of human ARSA cDNA under the control of the cytomegalovirus/b-actin hybrid (CAG) promoter in 6-month-old MLD mice that already show marked sulfatide accumulation and brain pathology. Within 3 months, a single intravenous injection of AAVPHP.eB-hARSA-HA resulted in correction of brain and spinal cord sulfatide storage, and improvement of astrogliosis and microgliosis in brain and spinal cord of treated animals. These results strongly support to consider the use of AAVPHP.eB-hARSA vector for intravenous gene therapy in symptomatic rapidly progressing forms of MLD.

Sustained phenotypic correction of hemophilia B dogs with a factor IX null mutation by liver-directed gene therapy

Blood ◽  
2002 ◽  
Vol 99 (8) ◽  
pp. 2670-2676 ◽  
Author(s):  
Jane D. Mount ◽  
Roland W. Herzog ◽  
D. Michael Tillson ◽  
Susan A. Goodman ◽  
Nancy Robinson ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Hemophilia B ◽  
Large Animal Model ◽  
Null Mutation ◽  
Large Animal ◽  
Adeno Associated Virus ◽  
Increased Risk

Abstract Hemophilia B is an X-linked coagulopathy caused by absence of functional coagulation factor IX (FIX). Using adeno-associated virus (AAV)–mediated, liver-directed gene therapy, we achieved long-term (> 17 months) substantial correction of canine hemophilia B in 3 of 4 animals, including 2 dogs with an FIX null mutation. This was accomplished with a comparatively low dose of 1 × 1012 vector genomes/kg. Canine FIX (cFIX) levels rose to 5% to 12% of normal, high enough to result in nearly complete phenotypic correction of the disease. Activated clotting times and whole blood clotting times were normalized, activated partial thromboplastin times were substantially reduced, and anti-cFIX was not detected. The fourth animal, also a null mutation dog, showed transient expression (4 weeks), but subsequently developed neutralizing anti-cFIX (inhibitor). Previous work in the canine null mutation model has invariably resulted in inhibitor formation following treatment by either gene or protein replacement therapies. This study demonstrates that hepatic AAV gene transfer can result in sustained therapeutic expression in a large animal model characterized by increased risk of a neutralizing anti-FIX response.

scholarly journals Gene therapy with hematopoietic stem and progenitor cell for monogenic disorders: a systematic review and meta-analysis

2021 ◽  
Author(s):  
Francesca Tucci ◽  
Stefania Galimberti ◽  
Luigi Naldini ◽  
Maria G Valsecchi ◽  
Alessandro Aiuti
Keyword(s):  
Gene Therapy ◽  
Metabolic Diseases ◽  
Ex Vivo ◽  
Meta Analysis ◽  
Primary Immunodeficiencies ◽  
Hematopoietic Stem ◽  
Monogenic Disorders ◽  
Monogenic Diseases ◽  
Stem And Progenitor Cells ◽  
Ex Vivo Gene Therapy

Abstract To provide an assessment of the safety of ex-vivo gene therapy (GT) with hematopoietic stem and progenitor cells (HSPC), we reviewed in a systematic manner the literature on monogenic diseases to describe survival, genotoxicity and engraftment of gene corrected HSPC, across vector platforms and diseases. From 1995 to 2020, 55 trials for 14 diseases met inclusion criteria and 406 patients with primary immunodeficiencies (55.2%), metabolic diseases (17.0%), haemoglobinopathies (24.4%) and bone marrow failures (3.4%) were treated with gammaretroviral vector (γRV) (29.1%), self-inactivating γRV (2.2%) or lentiviral vectors (LV) (68.7%). The pooled overall incidence rate of death was 0.9 per 100 person-years of observation (PYO) (95%CI = 0.37–2.17). There were 21 genotoxic events out of 1504.02 PYO. All these events occurred in γRV trials (0.99 events per 100 PYO, 95%CI = 0.18–5.43) for primary immunodeficiencies. Pooled rate of engraftment was 86.1% (95%CI = 66.9–95.0%) for γRV and 99.0% (95%CI = 95.1–99.8%) for LV HSPC-GT (p = 0.002). A comprehensive meta-analysis on HSPC-GT showed stable reconstitution of haematopoiesis in most recipients with superior engraftment and safer profile in patients receiving LV-transduced HSPC.

scholarly journals Gene therapy and viral vectors. Adeno-associated virus vectors and gene therapy.

Uirusu ◽  
1997 ◽  
Vol 47 (2) ◽  
pp. 221-230
Author(s):  
Masashi Urabe ◽  
Keiya Ozawa
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Adeno Associated Virus ◽  
Virus Vectors

scholarly journals Weighing the DNA content of Adeno-Associated Virus vectors with zeptogram precision using nanomechanical resonators

2021 ◽  
Author(s):  
Georgios Katsikis ◽  
Iris E Hwang ◽  
Wade Wang ◽  
Vikas S Bhat ◽  
Nicole L McIntosh ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Quality Control ◽  
Viral Vectors ◽  
Analytical Ultracentrifugation ◽  
Vaccine Development ◽  
Turnaround Time ◽  
Charge Detection ◽  
Adeno Associated Virus ◽  
Nanomechanical Resonators

Quantifying the composition of viral vectors used in vaccine development and gene therapy is critical for assessing their functionality. Adeno-Associated Virus (AAV) vectors, which are the most widely used viral vectors for in-vivo gene therapy, are typically characterized using PCR, ELISA, and Analytical Ultracentrifugation which require laborious protocols or hours of turnaround time. Emerging methods such as Charge-Detection Mass Spectroscopy, Static Light Scattering, and Mass Photometry offer turnaround times of minutes for measuring AAV mass, but mostly require purified AAV-based reference materials for calibration. Here, we demonstrate a method for using Suspended Nanomechanical Resonators (SNR) to directly measure both AAV mass and aggregation from a few microliters of sample within minutes. We achieve a resolution near 10 zeptograms which corresponds to 1% of the genome holding capacity of the AAV capsid. Our results show the potential of our method for providing real-time quality control of viral vectors during biomanufacturing.

Inactivation of a GFP retrovirus occurs at multiple levels in long-term repopulating stem cells and their differentiated progeny

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 894-901 ◽  
Author(s):  
Christopher A. Klug ◽  
Samuel Cheshier ◽  
Irving L. Weissman
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Gene Therapy ◽  
Stem Cell ◽  
Viral Vectors ◽  
Fluorescent Protein ◽  
Lymphoid Cells ◽  
Gfp Expression ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cell gene therapy holds promise for the treatment of many hematologic disorders. One major variable that has limited the overall success of gene therapy to date is the lack of sustained gene expression from viral vectors in transduced stem cell populations. To understand the basis for reduced gene expression at a single-cell level, we have used a murine retroviral vector, MFG, that expresses the green fluorescent protein (GFP) to transduce purified populations of long-term self-renewing hematopoietic stem cells (LT-HSC) isolated using the fluorescence-activated cell sorter. Limiting dilution reconstitution of lethally irradiated recipient mice with 100% transduced, GFP+ LT-HSC showed that silencing of gene expression occurred rapidly in most integration events at the LT-HSC level, irrespective of the initial levels of GFP expression. When inactivation occurred at the LT-HSC level, there was no GFP expression in any hematopoietic lineage clonally derived from silenced LT-HSC. Inactivation downstream of LT-HSC that stably expressed GFPin long-term reconstituted animals was restricted primarily to lymphoid cells. These observations suggest at least 2 distinct mechanisms of silencing retrovirally expressed genes in hematopoietic cells.

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