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![Green fluorescent protein (GFP) expression within nonhuman primate skeletal muscle. GFP immunofluorescence from adeno-associated virus type 9 (AAV9)-injected and phosphate-buffered saline (PBS)-injected monkeys demonstrates extensive transgene expression in skeletal muscles of all AAV9-injected animals. GFP expression was detected in the brachial limbs (triceps brachii a–c), trunk [diaphragm e–g and transverse abdominus (TVA) i–k], pelvic limbs (quadriceps m–o, gastrocnemius q–s, and tibialis anterior u–w) and head (tongue x–z) of AAV9 systemically injected animals. No GFP signal was detected in any of the muscle from the PBS-injected animals (d,h,l,p,t, and aa).](profile/Brian-Coley/publication/51542229/figure/fig4/AS:214374259138569@1428122214572/Green-fluorescent-protein-GFP-expression-within-nonhuman-primate-skeletal-muscle-GFP.png)
Green fluorescent protein (GFP) expression within nonhuman primate skeletal muscle. GFP immunofluorescence from adeno-associated virus type 9 (AAV9)-injected and phosphate-buffered saline (PBS)-injected monkeys demonstrates extensive transgene expression in skeletal muscles of all AAV9-injected animals. GFP expression was detected in the brachial limbs (triceps brachii a–c), trunk [diaphragm e–g and transverse abdominus (TVA) i–k], pelvic limbs (quadriceps m–o, gastrocnemius q–s, and tibialis anterior u–w) and head (tongue x–z) of AAV9 systemically injected animals. No GFP signal was detected in any of the muscle from the PBS-injected animals (d,h,l,p,t, and aa).
Source publication
Adeno-associated virus type 9 (AAV9) is a powerful tool for delivering genes throughout the central nervous system (CNS) following intravenous injection. Preclinical results in pediatric models of spinal muscular atrophy (SMA) and lysosomal storage disorders provide a compelling case for advancing AAV9 to the clinic. An important translational step...
Contexts in source publication
Context 1
... struc- tures such as thalamus (Figure 3a,c) and putamen (Figure 3k,l) were also GFP + but at a lower cell density. In contrast to the mice, primate brains of all ages had primarily glial transduc- tion with microglia and astrocytes being the most prominent cell types targeted (Supplementary Figure S4) as determined by colabeling with Iba-1 and GFAP (microglia and astrocyte marker, respectively) with neurons interspersed throughout. The scarcity of neuronal transduction was most striking in the hippocampus (Figure 3k,n) and dentate gyrus of the nonhu- man primates because, in mice, neurons of these regions were highly transduced in both neonate-and adult-treated animals. ...Context 2
... Therefore, we examined skeletal muscle of the AAV9-injected nonhuman primates for GFP expression. In the young monkeys (P1-P90), all skeletal muscles examined were positive for GFP using immunofluorescent detection (Figure 4) including triceps, diaphragm, transverse abdominus, quadriceps, gastrocnemius, tibialis anterior, and tongue. Skeletal muscles sampled were from the brachial and pelvic limbs, head, and trunk indicating a body- wide distribution. ...Similar publications
The development of gene therapies for the treatment of diseases of the central nervous system has been hindered by the limited availability of adeno-associated viruses (AAVs) that efficiently traverse the blood–brain barrier (BBB). Here, we report the rational design of AAV9 variants displaying cell-penetrating peptides on the viral capsid and the...
Citations
... are underway in the United States to investigate a wide variety of gene delivery vehicles for both gene replacement and genome editing therapeutic strategies. The gene therapy field has made significant advancements over the last few decades as there are now Food and Drug Administration (FDA) approved therapies for multiple genetically inherited disorders that were once considered untreatable (1)(2)(3)(4)(5)(6)(7)(8)(9). ...
... AAV vectors are being increasingly incorporated into the development of gene therapies for a wide variety of genetically inherited disorders due to their ability to efficiently transduce various cell types, including non-dividing cells with relatively minimal immunogenicity (39)(40)(41)(42). AAV9 has demonstrated broad tropism for a variety of organs including the CNS and is the gene delivery vehicle currently being used in the FDA-approved therapy for SMA (7). Despite the overall clinical success with this therapy, there remain challenges due to the occurrence of immunogenicity to natural serotypes such as AAV9. ...
Highly efficient adeno associated viruses (AAVs) targeting the central nervous system (CNS) are needed to deliver safe and effective therapies for inherited neurological disorders. The goal of this study was to compare the organ-specific transduction efficiencies of two AAV capsids across three different delivery routes. We compared AAV9-CBA-fLucYFP to AAV-DJ-CBA-fLucYFP using the following delivery routes in mice: intracerebroventricular (ICV) 1x1012 vg/kg, intrathecal (IT) 1x1012 vg/kg, and intravenous (IV) 1x1013 vg/kg body weight. Our evaluations revealed that following ICV and IT administrations, AAV-DJ demonstrated significantly increased vector genome (vg) uptake throughout the CNS as compared to AAV9. Through the IV route, AAV9 demonstrated significantly increased vg uptake in the CNS. However, significantly fewer vgs were detected in the off-target organs (kidney and liver) following administration of AAV-DJ using the IT and IV delivery routes as compared to AAV9. Distributions of vgs correlate well with transgene transcript levels, luciferase enzyme activities, and immunofluorescence detection of YFP. Overall, between the two vectors, AAV-DJ resulted in better targeting and expression in CNS tissues paired with de-targeting and reduced expression in liver and kidneys. Our findings support further examination of AAV-DJ as a gene therapy capsid for the treatment of neurological disorders.
... We chose to do an open injection technique to avoid any technical variability from ultrasound-guided injections (which are performed routinely in humans, but are not readily translatable to this fetal lamb model, which has multiple fetuses and a different placental architecture). Our findings support previous reports 12,30,31 of broad biodistribution of AAV vectors across the neuraxis and in other tissues, but raise important safety considerations, particularly due to the observation of maternal exposure and germline transduction in female fetuses. Although viral integration is low, these findings may be particularly concerning if AAV vectors are used to deliver genome editors. ...
... www.moleculartherapy.org Molecular Therapy: Methods & Clinical Development Vol. 32 June 2024previously described,31 but adapted for sheep plasma samples. Briefly, a 2 Â 10 10 vector genome per milliliter solution of AAV9 capsids in 1 mM carbonate buffer were coated into a 96-well titer plate and incubated overnight at 4 C. ...
Prenatal somatic cell gene therapy (PSCGT) could potentially treat severe, early-onset genetic disorders such as spinal muscular atrophy (SMA) or muscular dystrophy. Given the approval of adeno-associated virus serotype 9 (AAV9) vectors in infants with SMA by the U.S. Food and Drug Administration, we tested the safety and biodistribution of AAV9-GFP (clinical-grade and dose) in fetal lambs to understand safety and efficacy after umbilical vein or intracranial injection on embryonic day 75 (E75) . Umbilical vein injection led to widespread biodistribution of vector genomes in all examined lamb tissues and in maternal uteruses at harvest (E96 or E140; term = E150). There was robust GFP expression in brain, spinal cord, dorsal root ganglia (DRGs), without DRG toxicity and excellent transduction of diaphragm and quadriceps muscles. However, we found evidence of systemic toxicity (fetal growth restriction) and maternal exposure to the viral vector (transient elevation of total bilirubin and a trend toward elevation in anti-AAV9 antibodies). There were no antibodies against GFP in ewes or lambs. Analysis of fetal gonads demonstrated GFP expression in female (but not male) germ cells, with low levels of integration-specific reads, without integration in select proto-oncogenes. These results suggest potential therapeutic benefit of AAV9 PSCGT for neuromuscular disorders, but warrant caution for exposure of female germ cells.
... 72 However, this is not the only method for AAV introduction to the brain. Intraventricular, 73 intrathecal, 74 intravenous, 75 intraocular, 76 intraspinal 77 injection techniques, among others, have been used. Furthermore, rapid development of gene therapies has motivated creation of many viral capsid variants with differential tissue and organ specificities. ...
Purpose
Gene‐expression reporter systems, such as green fluorescent protein, have been instrumental to understanding biological processes in living organisms at organ system, tissue, cell, and molecular scales. More than 30 years of work on developing MRI‐visible gene‐expression reporter systems has resulted in a variety of clever application‐specific methods. However, these techniques have not yet been widely adopted, so a general‐purpose expression reporter is still required. Here, we demonstrate that the manganese ion transporter Zip14 is an in vivo MRI‐visible, flexible, and robust gene‐expression reporter to meet this need.
Methods
Plasmid constructs consisting of a cell type–specific promoter, gene coding for human Zip14, and a histology‐visible tag were packaged into adeno‐associated viruses. These viruses were intracranially injected into the mouse brain. Serial in vivo MRI was performed using a vendor‐supplied 3D‐MPRAGE sequence. No additional contrast agents were administered. Animals were sacrificed after the last imaging timepoint for immunohistological validation.
Results
Neuron‐specific overexpression of Zip14 produced substantial and long‐lasting changes in MRI contrast. Using appropriate viruses enabled both anterograde and retrograde neural tracing. Expression of Zip14 in astrocytes also enabled MRI of glia populations in the living mammalian brain.
Conclusions
The flexibility of this system as an MRI‐visible gene‐expression reporter will enable many applications of serial, high‐resolution imaging of gene expression for basic science and therapy development.
... Of relevance to many mitochondrial diseases, including MELAS, is the ability to target cells of the central nervous system (CNS), particularly motor neurons 71 . Successful delivery to the CNS has been demonstrated following intravenous injection of AAV9 in mice 72 , non-human primates 73,74 and humans 75,76 . However, targeting of motor neurons was most effective following early postnatal administration 77 . ...
Nuclease-mediated editing of heteroplasmic mitochondrial DNA (mtDNA) seeks to preferentially cleave and eliminate mutant mtDNA, leaving wild-type genomes to repopulate the cell and shift mtDNA heteroplasmy. Various technologies are available, but many suffer from limitations based on size and/or specificity. The use of ARCUS nucleases, derived from naturally occurring I-CreI, avoids these pitfalls due to their small size, single-component protein structure and high specificity resulting from a robust protein-engineering process. Here we describe the development of a mitochondrial-targeted ARCUS (mitoARCUS) nuclease designed to target one of the most common pathogenic mtDNA mutations, m.3243A>G. mitoARCUS robustly eliminated mutant mtDNA without cutting wild-type mtDNA, allowing for shifts in heteroplasmy and concomitant improvements in mitochondrial protein steady-state levels and respiration. In vivo efficacy was demonstrated using a m.3243A>G xenograft mouse model with mitoARCUS delivered systemically by adeno-associated virus. Together, these data support the development of mitoARCUS as an in vivo gene-editing therapeutic for m.3243A>G-associated diseases.
... AAV vectors have the ability to transduce a wide range of terminally differentiated or non-dividing cells, such as neurons, and can also selectively transduce astrocytes, oligodendrocytes, endothelial cells, photoreceptor cells in the retina, and dorsal root ganglia. [27][28][29][30] AAV is well suited for transgene delivery due to its ability to transpose both dividing and nondividing cells for long-term stable expression with minimal risk to the subject. ...
Stroke has been associated with devastating clinical outcomes, with current treatment strategies proving largely ineffective. Therefore, there is a need to explore alternative treatment options for addressing post‐stroke functional deficits. Gene therapy utilizing adeno‐associated viruses (AAVs) as a critical gene vector delivering genes to the central nervous system (CNS) gene delivery has emerged as a promising approach for treating various CNS diseases. This review aims to provide an overview of the biological characteristics of AAV vectors and the therapeutic advancements observed in preclinical models of ischemic stroke. The study further investigates the potential of manipulating AAV vectors in preclinical applications, emphasizing the challenges and prospects in the selection of viral vectors, drug delivery strategies, immune reactions, and clinical translation.
... 47 Efficient CNS targeting has also been demonstrated in large animals at different ages. 48 Importantly, AAV9 has been shown to target efficiently the PNS via intrathecal, 22,28,32 intravenous, 32 or intraneural administration, 49 with high degree of tropism for myelinating Schwann cells leading to therapeutic GJB1 gene expression controlled by the Schwann cell-specific Mpz promoter to treat CMT1X neuropathy, 22,32 or to PMP22 gene silencing by shRNA 49 or microRNA 28 under ubiquitous promoters. ...
Type 4C Charcot-Marie-Tooth (CMT4C) demyelinating neuropathy is caused by autosomal recessive SH3TC2 gene mutations. SH3TC2 is highly expressed in myelinating Schwann cells. CMT4C is a childhood-onset progressive disease without effective treatment. Here we generated a gene therapy for CMT4C mediated by an adeno-associated viral 9 vector (AAV9) to deliver the human SH3TC2 gene in the Sh3tc2-/- mouse model of CMT4C. We used a minimal fragment of the myelin protein zero (Mpz) promoter (miniMpz) which was cloned and validated to achieve Schwann cell-targeted expression of SH3TC2. Following the demonstration of AAV9-miniMpz.SH3TC2myc vector efficacy to re-establish SH3TC2 expression in the PNS, we performed an early as well as a delayed treatment trial in Sh3tc2-/- mice. We demonstrate both after early as well as following late treatment improvements in multiple motor performance tests and nerve conduction velocities. Moreover, treatment led to normalization of the organization of the nodes of Ranvier, which is typically deficient in CMT4C patients and Sh3tc2-/- mice, along with reduced ratios of demyelinated fibers, increased myelin thickness and reduced g-ratios at both time points of intervention. Taken together, our results provide a proof of concept for an effective and potentially translatable gene replacement therapy for CMT4C treatment.
... This time frame corresponds to the posttreatment MRI acquisition protocol to confirm successful BBB opening and discard abnormalities, such as edema, microhemorrhage, and infarction. The amount of AAV vectors injected was determined on the basis of previous reports on their intravenous infusions in macaque monkeys (27,36,54). After the infusion, the monkeys were monitored until full recovery from anesthesia. ...
Intracerebral vector delivery in nonhuman primates has been a major challenge. We report successful blood-brain barrier opening and focal delivery of adeno-associated virus serotype 9 vectors into brain regions involved in Parkinson's disease using low-intensity focus ultrasound in adult macaque monkeys. Openings were well tolerated with generally no associated abnormal magnetic resonance imaging signals. Neuronal green fluorescent protein expression was observed specifically in regions with confirmed blood-brain barrier opening. Similar blood-brain barrier openings were safely demonstrated in three patients with Parkinson's disease. In these patients and in one monkey, blood-brain barrier opening was followed by 18F-Choline uptake in the putamen and midbrain regions based on positron emission tomography. This indicates focal and cellular binding of molecules that otherwise would not enter the brain parenchyma. The less-invasive nature of this methodology could facilitate focal viral vector delivery for gene therapy and might allow early and repeated interventions to treat neurodegenerative disorders.
... For example, in a study aimed to investigate AAV1-9 distribution following systemic delivery in a murine model, AAV9 has shown rapid-onset, the best genome distribution, and the highest protein levels, in comparison with all other AAVs [92]. Moreover, in the CNS of murine, NHP, and feline models, it has a unique feature compared to other serotypes, as it can cross the BBB and transduce not only neuronal but also non-neuronal cells, including astrocytes, that cannot usually be transduced by other AAVs [27, [159][160][161][162], also showing tropism to photoreceptor cells in the retina [163]. Viral vectors based on AAV9 have also proven to be more efficient than those of AAV1 and AAV8 (in some cases 5-10-fold higher than AAV8), for murine, NHP, and porcine cardiac muscle transduction [164][165][166][167][168][169], enabled with another distinctive feature of AAV9-its ability to traverse the physical barrier of vascular system endothelia [168]. ...
... Similarly, spinal muscular atrophy (SMA) has always represented an attractive candidate for gene therapy, being caused by a single gene defect that affects the survival motor neuron (SMN) protein [235]. Therefore, SMA has been suggested as a target for AAV-mediated therapeutic approaches, mainly using AAV9 [161]. An open-label, phase I clinical trial was designed by AveXis, Inc. in 2014 to assess the safety and efficacy of AA9-SMN as a treatment for SMA [236]. ...
... Dual-ion-exchange chromatography, iodixanol gradient centrifugation murine, canine, and 115 hepatocytes [92,112,[141][142][143][144][145][146][147], murine skeletal and cardiac muscles [148], murine pancreatic cells [149,150], murine renal cells [151], and different cells in the murine retina [152][153][154] AAV9 terminal N-linked galactose putative integrin, LR glycosylation, ubiquitination, phosphorylation, SUMOylation, and acetylation Sucrose gradient centrifugation, and ion-exchange chromatography murine, NHP, and feline neuronal and non-neuronal cells, including astrocytes [27, [159][160][161][162], murine and NHP retinal photoreceptors cells [163], murine, NHP, and porcine cardiac muscle tissue [164][165][166][167][168][169], murine hepatocytes, skeletal muscles, and pancreatic cells [81,169], photoreceptor cells [170], renal tubular epithelium cells [171][172][173], Leydig cells in the testicular interstitial tissue [174], and alveolar and nasal epithelia [175,176]. ...
Despite scientific discoveries in the field of gene and cell therapy, some diseases still have no effective treatment. Advances in genetic engineering methods have enabled the development of effective gene therapy methods for various diseases based on adeno-associated viruses (AAVs). Today, many AAV-based gene therapy medications are being investigated in preclinical and clinical trials, and new ones are appearing on the market. In this article, we present a review of AAV discovery, properties, different serotypes, and tropism, and a following detailed explanation of their uses in gene therapy for disease of different organs and systems.
... It is worth noting that AAV-mediated delivery appears to be much more effective in mice 42 than larger animals like non-human primates 43,44,45 -perhaps due to differences in extravasation. Thus, we may need to use my aforementioned delivery system to effectively deliver a gene vector encoding hTERT in humans in a manner I described in my anti-aging article. ...
Aging kills 100,000 people a day - more than any other cause of death combined. The exact causes of aging have been much discussed, but the most pressing issue with regard to aging appears to me to be lipofuscin accumulation. That is, the accumulation of indigestible cellular garbage that needs to be removed from our cells, then the body. In this piece, I will explain why I think “getting rid of the garbage” should be at least one of our main goals with regard to longevity research for now.
... It is worth noting that AAV-mediated delivery appears to be much more effective in mice 42 than larger animals like non-human primates 43,44,45 -perhaps due to differences in extravasation. Thus, we may need to use my aforementioned delivery system to effectively deliver a gene vector encoding hTERT in humans in a manner I described in my anti-aging article. ...
Aging kills 100,000 people a day - more than any other cause of death combined. The exact causes of aging have been much discussed, but the most pressing issue with regard to aging appears to me to be lipofuscin accumulation. That is, the accumulation of indigestible cellular garbage that needs to be removed from our cells, then the body. In this piece, I will explain why I think “getting rid of the garbage” should be at least one of our main goals with regard to longevity research for now.
