Mucopolysaccharidosis kind I (MPS I) is an inherited metabolic dysfunction brought on by deficiency of the lysosomal enzyme alpha-L-iduronidase (IDUA). The 2 present therapies [hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT)], are insufficiently efficient in addressing neurologic illness, partially as a result of lack of ability of lysosomal enzyme to cross the blood mind barrier.
With a purpose to extra successfully deal with neurologic illness, we have now investigated the effectiveness of AAV-mediated IDUA gene supply to the mind utilizing a number of totally different routes of administration. Animals have been handled by both direct intracerebroventricular (ICV) injection, by intrathecal (IT) infusion into the cerebrospinal fluid, or by intranasal (IN) instillation of AAV9-IDUA vector.
AAV9-IDUA was administered to IDUA-deficient mice that have been both immunosuppressed with cyclophosphamide (CP), or immunotolerized at beginning by weekly injections of human iduronidase. In animals handled by ICV or IT administration, ranges of IDUA enzyme ranged from 3- to 1000-fold that of untamed kind ranges in all components of the microdissected mind.
In animals administered vector intranasally, enzyme ranges have been 100-fold that of untamed kind within the olfactory bulb, however enzyme expression was near wild kind ranges in different components of the mind.
Glycosaminoglycan ranges have been decreased to regular in ICV and IT handled mice, and in IN handled mice they have been normalized within the olfactory bulb, or decreased in different components of the mind.
Immunohistochemical evaluation confirmed in depth IDUA expression in all components of the mind of ICV handled mice, whereas IT handled animals confirmed transduction that was primarily restricted to the hind mind with some sporadic labeling seen within the mid- and fore mind.
At 6 months of age, animals have been examined for spatial navigation, reminiscence, and neurocognitive operate within the Barnes maze; all handled animals have been indistinguishable from regular heterozygous management animals, whereas untreated IDUA poor animals exhibited vital studying and spatial navigation deficits.
We conclude that IT and IN routes are acceptable and alternate routes of administration, respectively, of AAV vector supply to the mind with efficient IDUA expression, whereas all three routes of administration stop the emergence of neurocognitive deficiency in a mouse MPS I mannequin.
AAV9 transduction mediated by systemic supply of vector by way of retro-orbital injection in new child, neonatal and juvenile mice
Adeno-associated virus (AAV)-based gene remedy is gaining reputation owing to its glorious security profile and efficient therapeutic outcomes in quite a few ailments. Intravenous (IV) injection of AAV into the tail vein, facial vein and retro-orbital (RO) venous sinus have all been helpful methods to infuse the viral vector systemically.
Nevertheless, tail vein injection is technically difficult in juvenile mice, and injection at younger ages (≤postnatal day-(P)21) is actually unattainable. The temporal or facial vein is localized anterior to the ear bud and is markedly seen within the first couple of days postnatally.
Nevertheless, this technique is age-dependent and requires a dissecting microscope. Retro-orbital injection (ROI), however, is appropriate for all murine ages, together with new child and older mice, and is comparatively much less irritating to animals in comparison with tail vein injection.
Though many stories have proven ROI as an efficient route of AAV supply, herein we goal to spotlight and summarize the strategies and advantages of ROI. To seize the complete spectrum of transduction effectivity mediated by ROI, we transduced the editing-dependent reporter mice (Ai9 Cre reporter mice) with the AAV9 vector, which targets a variety of peripheral tissues with distinctive mind tropism.
We additionally present a complete description of the ROI method to facilitate viral vector administration with out problems.
Systemic Remedy of Fabry Illness Utilizing a Novel AAV9 Vector Expressing α-Galactosidase A
Fabry illness is a uncommon X-linked dysfunction affecting α-galactosidase A, a rate-limiting enzyme in lysosomal catabolism of glycosphingolipids. Present therapies current essential limitations, corresponding to low half-life and restricted distribution, which gene remedy can overcome.
The goal of this work was to check a novel adeno-associated viral vector, serotype 9 (AAV9), ubiquitously expressing human α-galactosidase A to deal with Fabry illness (scAAV9-PGK-GLA). The vector was preliminary examined in newborns of a Fabry illness mouse mannequin.
5 months after remedy, α-galactosidase A exercise was detectable within the analyzed tissues, together with the central nervous system. Furthermore, we examined the vector in grownup animals of each sexes at two doses and illness phases (presymptomatic and symptomatic) by single intravenous injection.
We discovered that the exogenous α-galactosidase A was energetic in peripheral tissues in addition to the central nervous system and prevented glycosphingolipid accumulation in handled animals as much as 5 months following injection. Antibodies towards α-galactosidase A have been produced in 9 out of 32 handled animals, though enzyme exercise in tissues was not considerably affected.
These outcomes display that scAAV9-PGK-GLA can drive widespread and sustained expression of α-galactosidase A, cross the blood mind barrier after systemic supply, and scale back pathological indicators of the Fabry illness mouse mannequin.
Single and twin vector gene remedy with AAV9-PHP.B rescues listening to in Tmc1 mutant mice
AAV-mediated gene remedy is a promising method for treating genetic listening to loss. Alternative or enhancing of the Tmc1 gene, encoding hair cell mechanosensory ion channels, is efficient for listening to restoration in mice with some limitations.
Environment friendly rescue of outer hair cell operate, in addition to lack of listening to restoration with later stage remedy, stay points to be solved. Exogenous genes delivered with the AAV9-PHP.B capsid by way of the utricle transduce each inside and outer hair cells of the mouse cochlea with excessive efficacy.
Right here we display that AAV9-PHP.B gene remedy can promote hair cell survival and efficiently rescues listening to in three distinct mouse fashions of listening to loss. Tmc1 alternative with AAV9-PHP.B in a Tmc1 knockout mouse rescues listening to and promotes hair cell survival with equal efficacy in inside and outer hair cells.
The identical remedy in a recessive Tmc1 listening to loss mannequin, Baringo, partially recovers listening to even with later stage remedy. Lastly, twin supply of SpCas9 and gRNA in separate AAV9-PHP.
AAVS1 Safe Harbor Targeting Vector 2.0 - GOI Knock-in Donor (AAVS1-SA-puro-EF1-MCS) |
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| GE622A-1 | SBI | 10 ug | EUR 1128 |
AAVS1 Safe Harbor Targeting Vector 2.0 - Reporter Knock-in Donor (AAVS1-SA-puro-MCS-GFP) |
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| GE624A-1 | SBI | 10 ug | EUR 1128 |
AAVS1 Safe Harbor Targeting Vector 2.0 - All-Purpose Donor (AAVS1-SA-puro-MCS), Complete Kit with CAS601A-1 (Cas9 SmartNuclease AAVS1-gRNA Targeting Vector) and GE640PR-1 (Junction PCR Primer Mix to confirm AAVS1 integration site) |
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| GE620A-KIT | SBI | 1 kit | EUR 2036 |
AAVS1 Safe Harbor Targeting Vector 2.0 - GOI Knock-in Donor (AAVS1-SA-puro-EF1-MCS), Complete Kit with CAS601A-1 (Cas9 SmartNuclease AAVS1-gRNA Targeting Vector) and GE640PR-1 (Junction PCR Primer Mix to confirm AAVS1 integration site) |
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| GE622A-KIT | SBI | 1 kit | EUR 2036 |
AAVS1 Safe Harbor Targeting Vector 2.0 - Reporter Knock-in Donor (AAVS1-SA-puro-MCS-GFP), Complete Kit with CAS601A-1 (Cas9 SmartNuclease AAVS1-gRNA Targeting Vector) and GE640PR-1 (Junction PCR Primer Mix to confirm AAVS1 integration site) |
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| GE624A-KIT | SBI | 1 kit | EUR 2036 |
pAAVK-EF1α -MCS AAVanced Cloning and Expression Vector |
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| AAV502A-1 | SBI | 10 ug | EUR 614 |
Cas9 SmartNuclease AAVS1-gRNA Targeting Vector [EF1a promoter] |
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| CAS601A-1 | SBI | 10 ug | EUR 996 |
pAAVK-EF1α -MCS-T2A-EGFP AAVanced Cloning and Expression Vector |
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| AAV526A-1 | SBI | 10 ug | EUR 614 |
pAAVK-EF1α -MCS-T2A-Puro AAVanced Cloning and Expression Vector |
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| AAV527A-1 | SBI | 10 ug | EUR 614 |
pAAVK-EF1α –MCS-T2A-mRFP AAVanced Cloning and Expression Vector |
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| AAV528A-1 | SBI | 10 ug | EUR 614 |
pAAVK-EF1α-MCS1-CMV-MCS2 AAVanced Cloning and Expression Vector |
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| AAV503A-1 | SBI | 10 ug | EUR 614 |
pAAVK-EF1α-MCS1-CMV-EGFP AAVanced Cloning and Expression Vector |
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| AAV536A-1 | SBI | 10 ug | EUR 614 |
pAAVK-EF1α-MCS1-CMV-Puro AAVanced Cloning and Expression Vector |
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| AAV537A-1 | SBI | 10 ug | EUR 614 |
pAAVK-EF1α-MCS1-CMV-mRFP AAVanced Cloning and Expression Vector |
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| AAV538A-1 | SBI | 10 ug | EUR 614 |
AAVS1 Safe Harbor cDNA/miRNA Donor Vector [pAAVS1D-PGK.MCS-EF1a.copGFPpuro] |
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| GE602A-1 | SBI | 10 ug | EUR 1190 |
PinPoint-HR attP Placement Vector – for targeting the Human AAVS1 Safe Harbor |
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| PIN410A-1 | SBI | 10ug | EUR 1190 |
5’ and 3’ AAVS1 Positive Control Donor Vector Primer Mixes for Junction PCR Assays (10 µM) |
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| GE603PR-1 | SBI | 100 ul | EUR 232 |
AAVS1 Safe Harbor Positive Control Donor Vector [pAAVS1D-CMV.RFP-EF1a.copGFPpuro] |
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| GE603A-1 | SBI | 10 ug | EUR 1190 |
AAV1 SaCas9 |
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| 78479 | BPS Bioscience | 50 µl x 2 | EUR 545 |
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Description: Adeno-Associated Virus Serotype 1 (AAV1) exhibits high homology with other AAV serotypes. AAV1 efficiently transduces muscle tissue, as determined by a region of the capsid protein VP1 (amino acids 350 to 430) which functions as a major determinant of tissue tropism._x000D_Cas9 is an endonuclease enzyme that introduces a double stranded break into the DNA when recruited to a specific DNA sequence by the sgRNA (single guide RNA). This double stranded break is repaired in mammalian cells either through Non-Homologous End Joining or Homologous Recombination. Non-Homologous End Joining often results in the deletion or insertion of several base pairs at the cut site, which, when resulting in a frameshift, causes the functional inactivation of the gene._x000D_SaCas9 (Staphylococcus aureus CRISPR-associated protein 9) has demonstrated high cutting efficiency in mammalian cells, and its smaller size makes it ideal for packaging into AAV. SaCas9 recognizes a longer protospacer adjacent motif (PAM) site, 5'-NNGRRT-3', than the more traditional SpCas9 (Streptococcus pyogenes CRISPR-associated protein 9). These AAV particles constitutively express SaCas9 under the control of a CMV promoter. |
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AAV2 SaCas9 |
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| 78480 | BPS Bioscience | 50 µl x 2 | EUR 545 |
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Description: Adeno-Associated Virus serotype 2 (AAV2) is the best characterized AAV serotype. Nearly all recombinant AAV serotypes utilize the AAV2 inverted terminal repeats (ITRs). AAV2 requires the expression of Heparan Sulfate Proteoglycan (HSPG) on the surface of host for cells for binding and internalization. Of nearly all the discovered AAV serotypes, AAV2 has the best transduction efficiency in cell culture and is the best tool for in vitro studies._x000D_Cas9 is an endonuclease enzyme that introduces a double stranded break into the DNA when recruited to a specific DNA sequence by the sgRNA (single guide RNA). This double stranded break is repaired in mammalian cells either through Non-Homologous End Joining or Homologous Recombination. Non-Homologous End Joining often results in the deletion or insertion of several base pairs at the cut site, which, when resulting in a frameshift, causes the functional inactivation of the gene._x000D_SaCas9 (Staphylococcus aureus CRISPR-associated protein 9) has demonstrated high cutting efficiency in mammalian cells, and its smaller size makes it ideal for packaging into AAV. SaCas9 recognizes a longer protospacer adjacent motif (PAM) site, 5'-NNGRRT-3', than the more traditional SpCas9 (Streptococcus pyogenes CRISPR-associated protein 9). These AAV particles constitutively express SaCas9 under the control of a CMV promoter. The protein contains a C-terminus HA tag. |
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AAV3 SaCas9 |
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| 78481 | BPS Bioscience | 50 µl x 2 | EUR 545 |
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Description: Adeno-Associated Virus Serotype 3 (AAV3) shares 82% sequence homology with AAV2, and like AAV2, requires the Heparan Sulfate Proteoglycan (HSPG) receptor for cell attachment. AAV3 vectors transduce human liver cancer cells extremely efficiently because AAV3 utilizes the human Hepatocyte Growth Factor Receptor (hHGFR) as a co-receptor for viral entry, which is highly expressed in these cells. Both the extracellular and intracellular kinase domains of hHGFR are required for AAV3-mediated transgene expression._x000D_Cas9 is an endonuclease enzyme that introduces a double stranded break into the DNA when recruited to a specific DNA sequence by the sgRNA (single guide RNA). This double stranded break is repaired in mammalian cells either through Non-Homologous End Joining or Homologous Recombination. Non-Homologous End Joining often results in the deletion or insertion of several base pairs at the cut site, which, when resulting in a frameshift, causes the functional inactivation of the gene._x000D_SaCas9 (Staphylococcus aureus CRISPR-associated protein 9) has demonstrated high cutting efficiency in mammalian cells, and its smaller size makes it ideal for packaging into AAV. SaCas9 recognizes a longer protospacer adjacent motif (PAM) site, 5'-NNGRRT-3', than the more traditional SpCas9 (Streptococcus pyogenes CRISPR-associated protein 9). These AAV particles constitutively express SaCas9 under the control of a CMV promoter. |
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AAV5 SaCas9 |
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| 78483 | BPS Bioscience | 50 µl x 2 | EUR 545 |
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Description: Adeno-Associated Virus Serotype 5 (AAV5) differs from other parvovirus serotypes according to serological and DNA hybridization data, and AAV5 also uses different inverted terminal repeats (ITRs) compared to other AAV serotypes. AAV5 is the most efficient vector for transducing sensory neurons and is good at mediating gene transfer into human and murine airway epithelia. In addition, AAV5 vectors show a higher tropism for both mouse and human dendritic cells than AAV1, AAV2, AAV7, or AAV8._x000D_Cas9 is an endonuclease enzyme that introduces a double stranded break into the DNA when recruited to a specific DNA sequence by the sgRNA (single guide RNA). This double stranded break is repaired in mammalian cells either through Non-Homologous End Joining or Homologous Recombination. Non-Homologous End Joining often results in the deletion or insertion of several base pairs at the cut site, which, when resulting in a frameshift, causes the functional inactivation of the gene._x000D_SaCas9 (Staphylococcus aureus CRISPR-associated protein 9) has demonstrated high cutting efficiency in mammalian cells, and its smaller size makes it ideal for packaging into AAV. SaCas9 recognizes a longer protospacer adjacent motif (PAM) site, 5'-NNGRRT-3', than the more traditional SpCas9 (Streptococcus pyogenes CRISPR-associated protein 9). These AAV particles constitutively express SaCas9 under the control of a CMV promoter. |
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AAV6 SaCas9 |
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| 78484 | BPS Bioscience | 50 µl x 2 | EUR 545 |
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Description: Adeno-Associated Virus serotype 6 (AAV6) appears to be related to AAV1 by sequence analysis and shows the best transduction efficiency in pancreatic beta-cells compared to other AAV serotypes. AAV6 vectors are also particularly effective in the transduction of human prostate, breast, and liver cancer cells._x000D_Cas9 is an endonuclease enzyme that introduces a double stranded break into the DNA when recruited to a specific DNA sequence by the sgRNA (single guide RNA). This double stranded break is repaired in mammalian cells either through Non-Homologous End Joining or Homologous Recombination. Non-Homologous End Joining often results in the deletion or insertion of several base pairs at the cut site, which, when resulting in a frameshift, causes the functional inactivation of the gene._x000D_SaCas9 (Staphylococcus aureus CRISPR-associated protein 9) has demonstrated high cutting efficiency in mammalian cells, and its smaller size makes it ideal for packaging into AAV. SaCas9 recognizes a longer protospacer adjacent motif (PAM) site, 5'-NNGRRT-3', than the more traditional SpCas9 (Streptococcus pyogenes CRISPR-associated protein 9). These AAV particles constitutively express SaCas9 under the control of a CMV promoter. |
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AAV8 SaCas9 |
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| 78485 | BPS Bioscience | 50 µl x 2 | EUR 545 |
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Description: Adeno-Associated Virus serotype 8 (AAV8) was isolated from rhesus monkey tissue, and the AAV8 rep and cap nucleotide sequences have 88% homology with AAV7 and 82% with AAV2. AAV8 exhibits greater transduction efficiency in the liver than other AAV serotypes. AAV8 and 9 have recently been used to correct disease-causing mutations and improve muscle function in mouse models of Duchenne muscular dystrophy._x000D_Cas9 is an endonuclease enzyme that introduces a double stranded break into the DNA when recruited to a specific DNA sequence by the sgRNA (single guide RNA). This double stranded break is repaired in mammalian cells either through Non-Homologous End Joining or Homologous Recombination. Non-Homologous End Joining often results in the deletion or insertion of several base pairs at the cut site, which, when resulting in a frameshift, causes the functional inactivation of the gene._x000D_SaCas9 (Staphylococcus aureus CRISPR-associated protein 9) has demonstrated high cutting efficiency in mammalian cells, and its smaller size makes it ideal for packaging into AAV. SaCas9 recognizes a longer protospacer adjacent motif (PAM) site, 5'-NNGRRT-3', than the more traditional SpCas9 (Streptococcus pyogenes CRISPR-associated protein 9). These AAV particles constitutively express SaCas9 under the control of a CMV promoter. |
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AAV1 ELISA Kit |
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| 55R-PROPRAAV1 | Fitzgerald | 96 tests | EUR 1406.4 |
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Description: ELISA kit for detection of AAV1 in the research laboratory |
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AAV5 ELISA Kit |
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| 55R-PROPRAAV5 | Fitzgerald | 96 tests | EUR 1442.4 |
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Description: ELISA kit for detection of AAV5 in the research laboratory |
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AAV1 ZsGreen |
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| 78443 | BPS Bioscience | 50 µl x 2 | EUR 545 |
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Description: Adeno-Associated Virus Serotype 1 (AAV1) exhibits high homology with other AAV serotypes. AAV1 efficiently transduces muscle tissue, as determined by a region of the capsid protein VP1 (amino acids 350 to 430) which functions as a major determinant of tissue tropism._x000D_These AAV1 particles constitutively express ZsGreen under a CMV promoter. ZsGreen is a human codon-optimized variant of the green fluorescent protein isolated from reef coral (Zoanthus sp). It has been engineered for higher expression in mammalian cells and is up to four times brighter than enhanced GFP (eGFP). ZsGreen expression and AAV1 transduction efficiency can easily be verified and optimized by fluorescence microscopy or flow cytometry. ZsGreen has an excitation wavelength of 493 nm and an emission wavelength of 505 nm. |
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AAV2 ZsGreen |
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| 78444 | BPS Bioscience | 50 µl x 2 | EUR 545 |
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Description: Adeno-Associated Virus Serotype 2 (AAV2) is the best characterized AAV serotype. Nearly all recombinant AAV serotypes utilize the AAV2 inverted terminal repeats (ITRs). AAV2 requires the expression of Heparan Sulfate Proteoglycan (HSPG) on the surface of host cells for cell binding and internalization. Of nearly all the discovered AAV serotypes, AAV2 has the best transduction efficiency in cell culture and is the best tool for in vitro studies._x000D_These AAV2 particles constitutively express ZsGreen under a CMV promoter. ZsGreen is a human codon-optimized variant of the green fluorescent protein isolated from reef coral (Zoanthus sp). It has been engineered for higher expression in mammalian cells and is up to four times brighter than enhanced GFP (eGFP). ZsGreen expression and transduction efficiency can easily be verified and optimized by fluorescence microscopy or flow cytometry. ZsGreen has an excitation wavelength of 493 nm and an emission wavelength of 505 nm. |
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AAV3 ZsGreen |
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| 78445 | BPS Bioscience | 50 µl x 2 | EUR 545 |
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Description: Adeno-Associated Virus Serotype 3 (AAV3) shares 82% sequence homology with AAV2, and like AAV2, requires Heparan Sulfate Proteoglycan (HSPG) for cellular attachment. AAV3 vectors transduce human liver cancer cells extremely efficiently because AAV3 utilizes the human Hepatocyte Growth Factor Receptor (hHGFR) as a co-receptor for viral entry, which is highly expressed in these cells. Both the extracellular domain and the intracellular kinase domain of hHGFR are required for AAV3-mediated transgene expression._x000D_These AAV3 particles constitutively express ZsGreen under a CMV promoter. ZsGreen is a human codon-optimized variant of the green fluorescent protein isolated from reef coral (Zoanthus sp). It has been engineered for higher expression in mammalian cells and is up to four times brighter than enhanced GFP (eGFP). ZsGreen expression and AAV3 transduction efficiency can easily be verified and optimized by fluorescence microscopy or flow cytometry. ZsGreen has an excitation wavelength of 493 nm and an emission wavelength of 505 nm. |
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AAV4 ZsGreen |
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| 78446 | BPS Bioscience | 50 µl x 2 | EUR 545 |
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Description: Adeno-Associated Virus Serotype 4 (AAV4) contains an expanded p5 promoter region compared to either AAV2 or AAV3. The AAV4 Rep gene product shows greater than 90% homology with the Rep products of AAV2 and AAV3. AAV4 can transduce human, monkey, and rat cells, and efficiently transduces type B astrocytes in the subventricular zone and glia overlying the rostral migratory stream neural tube._x000D_These AAV particles constitutively express ZsGreen under a CMV promoter. ZsGreen is a human codon-optimized variant of the green fluorescent protein isolated from reef coral (Zoanthus sp). It has been engineered for higher expression in mammalian cells and is up to four times brighter than enhanced GFP (eGFP). ZsGreen expression and AAV transduction efficiency can easily be verified and optimized by fluorescence microscopy or flow cytometry. ZsGreen has an excitation wavelength of 493 nm and an emission wavelength of 505 nm. |
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B vectors selectively disrupts a dominant Tmc1 allele and preserves listening to in Beethoven mice, a mannequin of dominant, progressive listening to loss. Tmc1-targeted gene therapies utilizing single or twin AAV9-PHP.B vectors supply potent and versatile approaches for treating dominant and recessive deafness.