scholarly journals Enhanced CNS transduction from AAV.PHP.eB infusion into the cisterna magna of older adult rats compared to AAV9

Gene Therapy ◽  
2021 ◽  
Author(s):  
Diptaman Chatterjee ◽  
David J. Marmion ◽  
Jodi L. McBride ◽  
Fredric P. Manfredsson ◽  
David Butler ◽  
...  
Keyword(s):  
High Efficiency ◽  
Brain Regions ◽  
Adult Rats ◽  
Variable Effect ◽  
Cisterna Magna ◽  
Gene Therapies ◽  
Clinical Investigations ◽  
Animal System ◽  
Old Rats ◽  
Cns Targeting

AbstractThe development of high efficiency, central nervous system (CNS) targeting AAV-based gene therapies is necessary to address challenges in both pre-clinical and clinical investigations. The engineered capsids, AAV.PHP.B and AAV.PHP.eB, show vastly improved blood-brain barrier penetration compared to their parent serotype, AAV9, but with variable effect depending on animal system, strain, and delivery route. As most characterizations of AAV.PHP variants have been performed in mice, it is currently unknown whether AAV.PHP variants improve CNS targeting when delivered intrathecally in rats. We evaluated the comparative transduction efficiencies of equititer doses (6 × 1011vg) of AAV.PHP.eB-CAG-GFP and AAV9-CAG-GFP when delivered into the cisterna magna of 6–9-month old rats. Using both quantitative and qualitative assessments, we observed consistently superior biodistribution of GFP+ cells and fibers in animals treated with AAV.PHP.eB compared to those treated with AAV9. Enhanced GFP signal was uniformly observed throughout rostrocaudal brain regions in AAV.PHP.eB-treated animals with matching GFP protein expression detected in the forebrain, midbrain, and cerebellum. Collectively, these data illustrate the benefit of intracisternal infusions of AAV.PHP.eB as an optimal system to distribute CNS gene therapies in preclinical investigations of rats, and may have important translational implications for the clinical CNS targeting.

scholarly journals Advances and Obstacles in Homology-Mediated Gene Editing of Hematopoietic Stem Cells

2021 ◽  
Vol 10 (3) ◽  
pp. 513
Author(s):  
Christi T. Salisbury-Ruf ◽  
Andre Larochelle
Keyword(s):  
Gene Editing ◽  
High Efficiency ◽  
Viral Vector ◽  
Ex Vivo ◽  
Blood Disorders ◽  
Hematopoietic Stem ◽  
Gene Therapies ◽  
Clinical Investigations ◽  
Stem And Progenitor Cells ◽  
Programmable Nucleases

Homology-directed gene editing of hematopoietic stem and progenitor cells (HSPCs) is a promising strategy for the treatment of inherited blood disorders, obviating many of the limitations associated with viral vector-mediated gene therapies. The use of CRISPR/Cas9 or other programmable nucleases and improved methods of homology template delivery have enabled precise ex vivo gene editing. These transformative advances have also highlighted technical challenges to achieve high-efficiency gene editing in HSPCs for therapeutic applications. In this review, we discuss recent pre-clinical investigations utilizing homology-mediated gene editing in HSPCs and highlight various strategies to improve editing efficiency in these cells.

scholarly journals A Single Cisterna Magna Injection of AAV Leads to Binaural Transduction in Mice

2022 ◽  
Vol 9 ◽  
Author(s):  
Fabian Blanc ◽  
Alexis-Pierre Bemelmans ◽  
Corentin Affortit ◽  
Charlène Joséphine ◽  
Jean-Luc Puel ◽  
...  
Keyword(s):  
Inner Ear ◽  
High Efficiency ◽  
Spiral Ganglion ◽  
Cisterna Magna ◽  
Gene Therapies ◽  
Large Numbers ◽  
Delivery Routes ◽  
Basal Portion ◽  
Ganglion Neurons ◽  
Almost All

Viral-mediated gene augmentation, silencing, or editing offers tremendous promise for the treatment of inherited and acquired deafness. Inner-ear gene therapies often require a safe, clinically useable and effective route of administration to target both ears, while avoiding damage to the delicate structures of the inner ear. Here, we examined the possibility of using a cisterna magna injection as a new cochlear local route for initiating binaural transduction by different serotypes of the adeno-associated virus (AAV2/8, AAV2/9, AAV2/Anc80L65). The results were compared with those following canalostomy injection, one of the existing standard inner ear local delivery routes. Our results demonstrated that a single injection of AAVs enables high-efficiency binaural transduction of almost all inner hair cells with a basal-apical pattern and of large numbers of spiral ganglion neurons of the basal portion of the cochlea, without affecting auditory function and cochlear structures. Taken together, these results reveal the potential for using a cisterna magna injection as a local route for binaural gene therapy applications, but extensive testing will be required before translation beyond mouse models.

Homeostatic control of manganese excretion in the neonatal rat

1987 ◽  
Vol 252 (5) ◽  
pp. R842-R847 ◽  
Author(s):  
N. Ballatori ◽  
E. Miles ◽  
T. W. Clarkson
Keyword(s):  
Trace Metal ◽  
Biliary Excretion ◽  
Intraperitoneal Injection ◽  
Neonatal Rat ◽  
Abrupt Increase ◽  
Adult Rats ◽  
Tracer Dose ◽  
Diminished Capacity ◽  
Carrier Free ◽  
Old Rats

Previous studies in neonatal and suckling animals showed that immature animals have a greatly diminished capacity to excrete manganese and therefore were considered to be unable to regulate tissue manganese concentrations. In contrast, the present studies indicate that suckling rats have the capacity to excrete excess manganese at rates nearly comparable to those of adults. Eight- to 10-day-old rats given a tracer dose of 54MnCl2 (essentially carrier free), either via gavage or by intraperitoneal injection showed little elimination of the 54Mn until the 18-19th day of life, when there was an abrupt increase in the rate of the metal's excretion. However, when manganese was given in doses of 1 and 10 mg/kg, the young animals excreted from 30-70% of the dose in only 4 days, at which time a new rate of excretion was achieved. This enhanced rate of excretion remained constant until the 18-19th day of life, when it was again accelerated. Biliary excretion of manganese, the primary route for the elimination of the metal, was only 30-60% lower in 14-day-old rats compared with adults at doses ranging from tracer to 10 mg 54Mn/kg. For both the 14-day-old and adult rats, an apparent biliary transport maximum was reached at a dose of 10 mg Mn/kg. These studies indicate that the excretory pathways for manganese are well developed in the neonatal rat. The avid retention of tracer quantities of manganese by the neonate may be a consequence of the scarcity of this essential trace metal in its diet.

scholarly journals The association of the sulphogalactosylglycerolipid of rat brain with myelination

1977 ◽  
Vol 166 (3) ◽  
pp. 421-428 ◽  
Author(s):  
Joanne Pieringer ◽  
G. Subba Rao ◽  
Paul Mandel ◽  
Ronald A. Pieringer
Keyword(s):  
Rat Brain ◽  
Adult Life ◽  
Developmental Pattern ◽  
Adult Rats ◽  
Jimpy Mouse ◽  
Myelin Fraction ◽  
Old Rats

The sulphogalactosylglycerolipid of rat brain is closely associated with the process of myelination, as demonstrated by the following observations. 1. The lipid is barely detectable in rat brain before 10 days of age, accumulates rapidly between age 10 and 25 days, and remains relatively constant in amount (between 0.3 and 0.4μmol per brain) thereafter into adult life. 2. The activity of adenosine 3′-phosphate 5′-sulphatophosphate–galactosyldiacylglycerol sulphotransferase is almost absent before 10 days of age, attains a maximum at age 20 days, and slowly decreases thereafter with increasing age. This developmental pattern correlates well with that of other myelin-specific metabolites. 3. Both the concentration of the sulphogalactosylglycerolipid and the activity of sulphotransferase are greatly decreased in the non-myelinating jimpy mouse. 4. The myelin fraction of rat brain contains most of the sulphogalactosylglycerolipid. The lipid occurs in a diacyl and an alkylacyl form. Determinations of the relative amount of each type in brain showed about a 1:1 mixture in both 21-day-old and adult rats. Rats injected with H235SO4 at 20 days of age lost35S from the diacyl form at a higher rate than from the alkylacyl compound over a 21-day period. These data suggest that the diacyl form has a higher turnover than the alkylacyl derivative. The percentage of the total sulpholipid content of brain contributed by the sulphogalactosylglycerolipid is 16% in 21-day-old rats and 8.4% in adult rats.

scholarly journals Differential effects of targeted tongue exercise and treadmill running on aging tongue muscle structure and contractile properties

2013 ◽  
Vol 114 (4) ◽  
pp. 472-481 ◽  
Author(s):  
Heidi Kletzien ◽  
John A. Russell ◽  
Glen E. Leverson ◽  
Nadine P. Connor
Keyword(s):  
Young Adult ◽  
Exercise Group ◽  
Contractile Properties ◽  
Treadmill Running ◽  
Brown Norway ◽  
Middle Aged ◽  
Adult Rats ◽  
Tongue Muscle ◽  
Muscle Structure ◽  
Old Rats

Age-associated changes in tongue muscle structure and strength may contribute to dysphagia in elderly people. Tongue exercise is a current treatment option. We hypothesized that targeted tongue exercise and nontargeted exercise that activates tongue muscles as a consequence of increased respiratory drive, such as treadmill running, are associated with different patterns of tongue muscle contraction and genioglossus (GG) muscle biochemistry. Thirty-one young adult, 34 middle-aged, and 37 old Fischer 344/Brown Norway rats received either targeted tongue exercise, treadmill running, or no exercise (5 days/wk for 8 wk). Protrusive tongue muscle contractile properties and myosin heavy chain (MHC) composition in the GG were examined at the end of 8 wk across groups. Significant age effects were found for maximal twitch and tetanic tension (greatest in young adult rats), MHCIIb (highest proportion in young adult rats), MHCIIx (highest proportion in middle-aged and old rats), and MHCI (highest proportion in old rats). The targeted tongue exercise group had the greatest maximal twitch tension and the highest proportion of MHCI. The treadmill running group had the shortest half-decay time, the lowest proportion of MHCIIa, and the highest proportion of MHCIIb. Fatigue was significantly less in the young adult treadmill running group and the old targeted tongue exercise group than in other groups. Thus, tongue muscle structure and contractile properties were affected by both targeted tongue exercise and treadmill running, but in different ways. Studies geared toward optimizing dose and manner of providing targeted and generalized tongue exercise may lead to alternative tongue exercise delivery strategies.

scholarly journals In vitro analysis of the origin and maintenance of O-2Aadult progenitor cells.

1992 ◽  
Vol 116 (1) ◽  
pp. 167-176 ◽  
Author(s):  
D Wren ◽  
G Wolswijk ◽  
M Noble
Keyword(s):  
Adult Life ◽  
Cell Types ◽  
Adult Rats ◽  
Optic Nerves ◽  
Limited Life ◽  
Old Rats ◽  
Vitro Analysis

We have been studying the differing characteristics of oligodendrocyte-type-2 astrocyte (O-2A) progenitors isolated from optic nerves of perinatal and adult rats. These two cell types display striking differences in their in vitro phenotypes. In addition, the O-2Aperinatal progenitor population appears to have a limited life-span in vivo, while O-2Aadult progenitors appear to be maintained throughout life. O-2Aperinatal progenitors seem to have largely disappeared from the optic nerve by 1 mo after birth, and are not detectable in cultures derived from optic nerves of adult rats. In contrast, O-2Aadult progenitors can first be isolated from optic nerves of 7-d-old rats and are still present in optic nerves of 1-yr-old rats. These observations raise two questions: (a) From what source do O-2Aadult progenitors originate; and (b) how is the O-2Aadult progenitor population maintained in the nerve throughout life? We now provide in vitro evidence indicating that O-2Aadult progenitors are derived directly from a subpopulation of O-2Aperinatal progenitors. We also provide evidence indicating that O-2Aadult progenitors are capable of prolonged self renewal in vitro. In addition, our data suggests that the in vitro generation of oligodendrocytes from O-2Aadult progenitors occurs primarily through asymmetric division and differentiation, in contrast with the self-extinguishing pattern of symmetric division and differentiation displayed by O-2Aperinatal progenitors in vitro. We suggest that O-2Aadult progenitors express at least some properties of stem cells and thus may be able to support the generation of both differentiated progeny cells as well as their own continued replenishment throughout adult life.

scholarly journals Phosphatidylcholine synthesis in the developing small intestine

1980 ◽  
Vol 186 (2) ◽  
pp. 399-403 ◽  
Author(s):  
P G Holtzapple ◽  
C M Starr ◽  
T Morck
Keyword(s):  
Small Intestine ◽  
Oleic Acid ◽  
Steady State ◽  
Adult Rats ◽  
Acyltransferase Activity ◽  
Acid Incorporation ◽  
Old Rats ◽  
Phosphatidylcholine Synthesis

1. Phosphatidylcholine synthesis in the foetal, newborn and adult small intestine of rats was studied by determination of cytidine diphosphocholine-1,2-diacylglycerocholine phosphotransferase (cholinephosphotransferase) and acyl-CoA-1-acyl-sn-glycerol-3-phosphocholine acyltransferase (lysophosphatidylcholine acyltransferase) activities and the incorporation of [1-14C]oleic acid into phosphatidylcholine. 2. Cholinephosphotransferase activity was low in foetal jejunum and ileum, increased 3-4 fold in the ileum by 6 days of age and by 12 days in the jejunum. Jejunal activity remained constant throughout weaning; ileal activity gradually decreased to values 25% of that of the jejunum. 3. Lysophosphatidylcholine acyltransferase activity was high in foetal jejunum and ileum, decreased 70% immediately after birth in the jejunum and increased to adult values by 12 days of age. Ileal activity decreased by 20% after birth, but decreased more rapidly at weaning to 30% of the activity in jejunum. 4. Initial rates and steady-state incorporation of [1-14C]oleic acid into phosphatidylcholine by jejunal rings of 10 day-old rats exceeded that observed in jejunal rings from adult rats by 2-4-fold. 5. In the postnatal jejunum, neither cholinephosphotransferase and lysophosphatidylcholine acyltransferase activities nor oleic acid incorporation were stimulated by cortisone administration in vivo.

Pituitary-adrenal response to bacterial endotoxin in developing rats

1988 ◽  
Vol 255 (4) ◽  
pp. E525-E530 ◽  
Author(s):  
L. Witek-Janusek
Keyword(s):  
Plasma Corticosterone ◽  
Neonatal Rat ◽  
Bacterial Endotoxin ◽  
Plasma Acth ◽  
Adult Rats ◽  
Age Related ◽  
Corticosterone Response ◽  
Adrenal Response ◽  
Old Rats ◽  
Related Pattern

The neonatal rat is very sensitive to the lethal effects of bacterial endotoxin. Because of the adaptive importance of pituitary-adrenal secretions to stress, this study examined the ontogeny of the plasma corticosterone and adrenocorticotropic hormone (ACTH) responses to endotoxin. The lethal sensitivity of young rats to endotoxin ranged from 0.5 to 30 mg/kg (ip) in the 1- to 21-day-old rat. After endotoxin treatment, the 1- and 2-day-old rat showed marked elevations of corticosterone similar in magnitude to that seen in 21-day-old and adult rats; however, significantly depressed corticosterone increments were observed in the 5-, 10-, and 14-day-old rats. This age-related pattern of adrenocortical secretion was correlated with the developing rat's corticosterone response to exogenous ACTH. In contrast, endotoxin administered to 5-, 10-, and 14-day-old rats resulted in increments of plasma ACTH similar to those observed in the 21-day-old and adult rats. Although plasma ACTH levels increased by 84-127% in the 1- and 2-day-old rats, these increases were significantly less than those of rats at all other ages tested. Thus the newborn rat mounts an effective corticosterone response to endotoxin, loses this ability between ages 5-14 days, and regains this response at 21 days of age. Because the hyporesponsive ages exhibit a marked increase in ACTH secretion, the loss of the adrenocortical response to endotoxin appears to be a result of a depressed responsiveness of the adrenal cortex to ACTH.

Increased NHE2 expression in rat intestinal epithelium during ontogeny is transcriptionally mediated

1998 ◽  
Vol 275 (4) ◽  
pp. C1143-C1150 ◽  
Author(s):  
James F. Collins ◽  
Pawel R. Kiela ◽  
Hua Xu ◽  
Jiamin Zeng ◽  
Fayez K. Ghishan
Keyword(s):  
Northern Blot ◽  
Membrane Vesicle ◽  
Mrna Levels ◽  
Rat Intestine ◽  
Functional Protein ◽  
Adult Rats ◽  
Specific Staining ◽  
Western Blots ◽  
Intestinal Membrane ◽  
Old Rats

We have previously described changes in intestinal brush-border membrane vesicle (BBMV) Na+/H+exchange activity and characterized Na+/H+exchanger (NHE3) expression during rat ontogeny. The current studies were designed to investigate developmental changes in NHE2 expression in rat intestine. In previous studies, pH-dependent uptake of Na+ in jejunal BBMV utilizing HOE-694 inhibition demonstrated that NHE2 functional protein levels were lowest in 2-wk-old rats, higher in 3-wk-old and adult rats, and highest in 6-wk-old rats [Collins et al. Am. J. Physiol. 273 ( Cell Physiol. 42): C1937–C1946, 1997]. In the current investigation, Northern blot analyses showed that NHE2 mRNA levels in the jejunum were similar in 6-wk-old, adult, and 3-wk-old rats and three- to fivefold lower in 2-wk-old rats. In situ hybridization of 2- and 6-wk-old rat intestine with NHE2-specific probes confirmed Northern blot observations. Polyclonal antibodies were developed against an NHE2-specific peptide from amino acids 652–661. Western blots with NHE2 antiserum showed that the intensity of a specific 90-kDa band was lowest in 2-wk-old animals and four- to sixfold higher in 3- and 6-wk-old and adult animals. Immunohistochemical analysis showed specific staining of NHE2 antiserum to only the apical intestinal membrane. Furthermore, nuclear run-on analyses showed a 1.7-fold higher NHE2 transcription rate in 6-wk-old rats than in 2-wk-old rats. Overall, the current data suggest that increases in NHE2 expression upon weaning are mediated by increased gene transcription.

Overexpression of PKC-βI and -δ contributes to higher PKC activity in the proximal tubules of old Fischer 344 rats

2003 ◽  
Vol 285 (6) ◽  
pp. F1100-F1107 ◽  
Author(s):  
Mohammad Asghar ◽  
Tahir Hussain ◽  
Mustafa F. Lokhandwala
Keyword(s):  
Skf 38393 ◽  
Proximal Tubules ◽  
Fischer 344 Rats ◽  
Adult Rats ◽  
Altered Expression ◽  
Fischer 344 ◽  
Pkc Isoforms ◽  
Diuretic Response ◽  
Old Rats ◽  
Pkc Ζ

Previously, we reported that natriuretic and diuretic response to dopamine is diminished in old Fischer 344 rats, which is due to higher basal protein kinase C (PKC) activity and hyperphosphorylation of Na-K-ATPase in the proximal tubules (PTs) of old rats. The present study was conducted to determine whether higher PKC activity could be due to altered expression of some of the PKC isoforms in the superficial cortex (rich in PTs) of old rats. Fluorimetric measurement showed almost twofold increase in the PKC activities in homogenates and membranes of old (24 mo) compared with adult (6 mo) rats. Interestingly, in the basal state PKC-βI was overexpressed in the membranes, whereas PKC-δ expression was increased in the cytosol of old compared with adult rats. Treatment of the cortical slices with either SKF-38393, a D1-like agonist, or PDBu, a direct activator of PKC, caused translocation of PKC-βI from cytosol to membranes in adult but not in old rats. Both of these drugs caused translocation of PKC-δ from membranes to cytosol in adult but not in old rats. These drugs had no effect on translocation of PKC-ζ in both adult and old rats. Both PKC-βI and -δ coimmunoprecipiated with α1-subunit of Na-K-ATPase in adult and old rats. These observations suggest that both SKF-38393 and PDBu differentially regulate PKC-βI and -δ in adult but not in old rats. Also, PKC-βI and -δ seem to interact with Na-K-ATPase in these animals. The overexpression of both PKC-βI and -δ in old rats could be responsible for a higher basal PKC activity, which causes the hyperphosphorylation of Na-K-ATPase and contributes to the diminished inhibition of Na-K-ATPase activity by dopamine in old rats.

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