ImmTOR nanoparticles enhance AAV-driven transgene expression after initial and repeat dosing in a mouse model of methylmalonic acidemia

Abstract To evaluate the consequences of the decline in mtDNA content associated with aging we have created an inducible mouse model expressing, in the polymerase domain of POLG1, a dominant-negative mutation that induces depletion of mtDNA. We utilized this inducible mouse model to modulate mitochondrial function by depleting and repleting the mtDNA content. We demonstrate that, in mice, ubiquitous expression of dominant-negative mutant POLG1 leads to 1) reduction of mtDNA content in skin, 2) skin wrinkles, and 3) hair loss. By turning off the mutant POLG1 transgene expression in the whole animal, the skin and hair phenotypes revert to normal after repletion of mtDNA. Thus, we have developed whole-animal mtDNA depleter-repleter mice. These mice present evidence that mtDNA homeostasis is involved in skin aging phenotype and loss of hair and provide an unprecedented opportunity to create tissue-specific mitochondrial modulation to determine the role of the mitochondria in a particular tissue.

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Robust Factor IX Expression Following ZFN-Mediated Genome Editing in An Adult Mouse Model of Hemophilia B

Blood ◽

10.1182/blood.v118.21.668.668 ◽

2011 ◽

Vol 118 (21) ◽

pp. 668-668

Author(s):

Xavier M Anguela ◽

Rajiv Sharma ◽

Hojun Li ◽

Virginia Haurigot ◽

Anand Bhagwat ◽

...

Keyword(s):

Mouse Model ◽

Genome Editing ◽

Partial Hepatectomy ◽

Transgene Expression ◽

Target Site ◽

Factor Ix ◽

Regulatory Control ◽

Neonatal Mice ◽

Proliferation Of Hepatocytes

Abstract Abstract 668 As a therapeutic strategy, site-specific modification of the genome has the potential to avoid some of the disadvantages of traditional gene replacement approaches such as insertional mutagenesis and lack of endogenous regulatory control of expression. We have recently reported that zinc finger nuclease (ZFN) driven gene correction can be achieved in vivo in a neonatal mouse model of hemophilia by combining AAV-mediated delivery of both the ZFNs and a Factor IX donor template with homology to the targeted F.IX gene (Li et al., Nature, 2011). The mouse model carries a mutant human F.IX mini-gene (hF9mut) knocked into the ROSA26 locus and ZFN-mediated cleavage followed by donor-dependent repair results in restoration of functional F.IX expression. AAV-ZFN and AAV-Donor vectors were administered to neonatal mice, where the rapid proliferation of hepatocytes in the growing animal may promote genome editing through homology directed repair (HDR). Here we sought to investigate whether ZFN-mediated genome editing is feasible in adult animals with predominantly quiescent hepatocytes. Tail vein injection of the AAV-ZFN and AAV-Donor, containing a promoterless wild type factor IX insert flanked by arms of homology to the target site, into adult (8 week old) mice (n=17) resulted in stable (>10wk) circulating F.IX levels of 730–1900 ng/mL (15-38% of normal), whereas mice receiving ZFN alone (n=9) exhibited F.IX levels below detection (<15 ng/mL). Co-delivery of AAV-Mock (luciferase expressing) & AAV-Donor (n=9), yielded <65 ng/mL F.IX. Importantly, mice lacking the hF9mut gene averaged less than 100 ng/mL after receiving AAV-ZFN and AAV-Donor (n=8), suggesting that F.IX expression was derived from on-target genome editing. To eliminate the potential for hF.IX expression resulting from episomal (non-integrated) AAV genomes we performed a two-thirds partial hepatectomy two days after AAV administration. Liver regeneration following hepatectomy is known to substantially reduce expression from non-integrated AAV genomes yet no significant differences in transgene expression were observed compared to non-hepatectomized mice: circulating F.IX levels in the AAV-ZFN + AAV-Donor group (n=13) ranged between 678–1240 ng/mL, whereas mice receiving ZFN alone (n=8) or Mock + AAV-Donor (n=8) had no detectable F.IX expression, or <100 ng/mL F.IX, respectively. Taken together, these data suggest that the F.IX expression in ZFN + Donor treated mice was derived from stable correction of the genome at the intended target site. In summary, we have shown that synchronized cell proliferation of hepatocytes, either in neonatal mice or following partial hepatectomy, is not necessary to achieve highly efficient genome editing and resultant high levels of transgene expression in vivo. These findings substantially expand the potential of ZFN-mediated genome editing as a therapeutic modality. Disclosures: Doyon: Sangamo Biosciences: Employment. Gregory:Sangamo Biosciences: Employment. Holmes:Sangamo Biosciences: Employment.

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359. Targeted Genome Editing Using TALENs to Correct a Mouse Model of Methylmalonic Acidemia (MMA)

Molecular Therapy ◽

10.1016/s1525-0016(16)33168-9 ◽

2016 ◽

Vol 24 ◽

pp. S144

Author(s):

Jessica L. Schneller ◽

Randy J. Chandler ◽

Charles P. Venditti

Keyword(s):

Mouse Model ◽

Genome Editing ◽

Methylmalonic Acidemia

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Targeted Integration of a CAR at a Novel Genomic Safe Harbor Directs Potent Therapeutic Outcomes

Blood ◽

10.1182/blood-2020-141967 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 28-28

Author(s):

Ashlesha Odak ◽

Han Yuan ◽

Judith Feucht ◽

Jorge Mansilla - Soto ◽

Justin Eyquem ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mouse Model ◽

Research Funding ◽

Transgene Expression ◽

T Cell Therapy ◽

Car T Cell ◽

Therapeutic Outcomes ◽

Car T Cell Therapy ◽

Car T

Chimeric receptor antigen (CAR)-T cell therapy using CARs specific for CD19 have been remarkably successful for treating chemo-refractory/relapsed B cell malignancies. These successes not withstanding, therapeutic outcomes between patients are variable and occasional cases of clonal expansion of the transduced T cells have been observed, albeit without leukemic transformation. These issues are cause for concern and need to be addressed to achieve better and safer therapeutic outcomes. Clonal expansion due to insertional mutagenesis and variegated transgene expression due to position effects are well established to be the consequence of the semi-random integration pattern afforded by gamma-retroviral and lentiviral vectors. We previously established that integration of a CAR cDNA in the TCR alpha locus (TRAC) provides consistent, regulated expression of CD19 CARs and superior CAR T cell efficacy in a mouse model of B-cell acute lymphoblastic leukemia (B-ALL). Here, we identify a novel extragenic site devoid of any known function and remote from endogenous genes, i.e. a 'genomic safe harbor' (GSH), that can be efficiently targeted in human T cells and drives potent CAR T cell therapy in the B-ALL mouse model. To identify GSHs that could be efficiently targeted in T cells by CRISPR-Cas9 and that could also support durable transgene expression, we screened for genomic regions meeting both, GSH criteria and high chromatin accessibility in T cells as measured by ATAC-seq. In human primary T cells, we identified 379 such sites. Ten of the highest accessible sites were investigated. All showed high (>90%) cleavage efficiency and allowed for CAR cDNA targeted integration and expression which also translated into effective cytolytic activity of the CARs within a few days after transduction. However, thereafter CAR expression diminished over the course of a week at most but not all of these sites. In order to prevent possible heterochromatinization, we incorporated chromatin insulator elements with barrier activity flanking the CAR transcription unit. Incorporation of the chromatin insulator element dramatically improved CAR expression and functionality at one site, whereas 3 other GSHs tested were not affected. One of the 10 GSHs maintained long-term CAR expression without requiring an insulator and directed potent anti-leukemic CAR T cell efficacy in a B-ALL 'CAR stress test' mouse model, matching the T cell potency afforded by integrating the CAR cDNA at the TRAC locus. This finding highlights the major effect of the integration site on transgene expression and ensuing therapeutic efficacy. We identified an extragenic GSH site that can be used for effective T cell engineering and sustained expression of a CAR. Through this study, we provide a platform for identifying GSHs that could be reliably targeted for safe and predictable expression of CARs or other immunomodulatory transgenes to potentiate adoptive immunotherapy. Disclosures Sadelain: Fate Therapeutics: Patents & Royalties, Research Funding; Mnemo: Patents & Royalties; Atara: Patents & Royalties, Research Funding; Takeda: Patents & Royalties, Research Funding; Minerva: Other: Biotechnologies, Patents & Royalties.

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168. Assaying Hepatic Correction Mediated by Varied AAV Vectors in a Knock-Out Transgenic Mouse Model of Methylmalonic Acidemia (MMA)

Molecular Therapy ◽

10.1016/s1525-0016(16)32977-x ◽

2016 ◽

Vol 24 ◽

pp. S65-S66

Author(s):

Brandon T. Hubbard ◽

Randy J. Chandler ◽

Charles P. Venditti

Keyword(s):

Mouse Model ◽

Transgenic Mouse ◽

Transgenic Mouse Model ◽

Methylmalonic Acidemia ◽

Knock Out

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Neurophysiological alterations in the nucleus reuniens of a mouse model of Alzheimer’s disease

10.1101/643023 ◽

2019 ◽

Author(s):

D.A. Walsh ◽

J.T. Brown ◽

A.D. Randall

Keyword(s):

Mouse Model ◽

Cortical Neurons ◽

Transgene Expression ◽

Membrane Properties ◽

List Type ◽

Nucleus Reuniens ◽

Model Of Alzheimer’S Disease ◽

Passive Membrane Properties ◽

Passive Membrane ◽

Rebound Firing

AbstractTransgenic mice that overproduce beta-amyloid (Aβ) peptides exhibit neurophysiological alterations at the cellular, synaptic and network levels. Recently, increased neuronal activity in nucleus reuniens (Re), has been linked to hyperexcitability within hippocampal-thalamo-cortical networks in the J20 mouse model of amyloidopathy. Here in vitro whole-cell patch clamp recordings were used to compare old pathology-bearing J20 mice and wild-type controls to examine whether alterations to the intrinsic electrophysiological properties of Re neurons could contribute to the amyloidopathy-associated Re hyperactivity. A greater proportion of Re neurons displayed a hyperpolarised membrane potential in J20 mice without changes to the incidence or frequency of spontaneous action potential (AP) generation. Passive membrane properties were independent of transgene expression. Re neurons recorded from J20 mice did not exhibit increased AP generation in response to depolarising current stimuli but did exhibit an increased propensity to rebound burst following hyperpolarising current stimuli. This increase in rebound firing does not appear to result from alterations to T-type calcium channels. Finally, in J20 mice there was an ∼8% reduction in spike width, similar to what we and others have reported in CA1 pyramidal neurons from multiple amyloidopathy mice. We conclude that alterations to the intrinsic properties of Re neurons may contribute to the hyperexcitability observed in hippocampal-thalmo-cortical circuits under pathological Aβ load.Key PointsAlterations in the neurophysiology of hippocampal and cortical neurons has been linked to network hyperexcitability in mouse models of amyloidopathy.The nucleus reuniens (Re) is part of a cognitive network involving the hippocampal formation and prefrontal cortex. Increased cellular activity in Re has been linked to the generation of hippocampal-thalamo-cortical seizure activity in J20 mice.Re neurons display hyperpolarised resting membrane potentials in J20 mice. Passive membrane properties are unaffected by transgene expression. Re neurons recorded from J20 mice did not exhibit increased excitability in response to depolarising current stimuli but did exhibit an increased propensity to rebound burst following hyperpolarising current stimuli. This increased rebound firing was not a result of changes in T-type Ca2+ conductances. Finally we observed a decrease in AP width.These results help us understand how altered Re cellular neurophysiology may contribute to hippocampal-thalamo-cortical hyperexcitability in J20 mice.

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Lentiviral Insertion Site Analysis of Corrected Mouse Model of β Thalassemia Following Lentiviral Transduction of Embryonic Stem Cells.

Blood ◽

10.1182/blood.v110.11.2592.2592 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2592-2592

Author(s):

Rui Yang ◽

Sean C. McConnell ◽

Yongliang Huo ◽

Clayton Ulrey ◽

Shan-Run Liu ◽

...

Keyword(s):

Stem Cells ◽

Mouse Model ◽

Peripheral Blood ◽

Transgene Expression ◽

Es Cells ◽

Globin Gene ◽

Embryonic Stem ◽

Lentiviral Transduction ◽

Insertion Sites ◽

Real Time Qpcr

Abstract The delivery of therapeutic transgenes via lentiviral transduction of stem cells holds great promise for future cell based therapies for inherited genetic disorders. The regulation of any transgene integrated into patient derived embryonic stem (ES) cells (generated via nuclear transfer or reprogramming) needs to be studied in great detail prior to their differentiation and transplantation back into the patient. Because genetically manipulated ES cells can be clonally isolated, expanded to great numbers in an undifferentiated state, and differentiated to specific cell types, they can be carefully tested in vitro prior to their reintroduction back into patients. ES cells derived from a mouse model of β thalassemia were transduced with a recombinant, self inactivating (SIN) lentiviral vector containing a 2.3 kb human β-globin gene and a 3.2 kb Locus Control Region composed of regulatory elements; HS2, HS3, and HS4. Clonal populations of transfected β thalassemic ES cells were isolated, archived, and utilized to produce genetically identical offspring by injection into tetraploid blastocysts or eight-cell embryos. Real-Time QPCR expression analyses demonstrated high levels of human β-globin gene expression in the peripheral blood of five of the six lines produced. The average expression level per transgene copy number ranged from 13% to 62% of endogenous mouse α-globin levels. Peripheral blood hemolysates analyzed by HPLC confirmed the high level production of human β-globin chains. Measurement of the red blood indices in these five lines showed that the anemia was corrected. In order to examine the influence of chromosomal position effects on individual transgene expression, the lentiviral insertion sites were mapped by linear amplification (LAM) PCR. Cloned mice from the six lines were bred in order to segregate each of their individual lentiviral transgenes. Offspring containing single insertion sites were analyzed for human β-globin transgene expression and synthesis by Real-Time QPCR of blood RNA and by HPLC of hemolysates, respectively. These analyses demonstrate that there is a preference for provirus integration in ES cells; all transgene insertions are not expressed equally; lentiviral transduction of a human β-globin gene into ES cells can cure a severe hemoglobinopathy; and that the delivery of therapeutic transgenes via lentiviral transduction of ES cells did not harm the developmental potential of the cells to produce an entire mouse.

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