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Take a deep breath and dive in. Sign up, take a look, free.porn and chat with others in the Singles on Cam community. In fact, she once turned down a career singing in order to cam chat porn. Laila Mickelwait, director of religious anti-trafficking organisation Exodus Cry, who posted the petition aimed at shutting down Pornhub. He had been joined by Nicole and their fellow judges Louis Walsh and Sharon Osbourne and according to The Sun, misjudged a corner while hunting down an ice cream. Some of these people does not look for sex in here, all they want to find new friends and spend their time while they get bored in office or home. In some embodiments, the subject has received ERT at baseline, while in other embodiments, the subject has not received ERT. In some embodiments, the reduced or delayed need for ERT is measured, for example, by a change from baseline or stabilization in total GAG, DS GAG, and HS GAG levels measured in liver tissue and CSF.

In some embodiments, the splice acceptor site (e.g. SA, SEQ ID NO:14) is derived, for example, from hF9 exon 2 to allow efficient splicing of the hIDS transcript, for example, into the mature mRNA from the albumin locus, and is effective with both types of the donor integration mechanisms (e.g. NHEJ or HDR). In some embodiments, provided herein is a method of reducing, stabilizing or eliminating urine GAGs (e.g. urine GAG levels) by treatment with the methods and compositions disclosed herein as compared with a subject that has not been treated, the method comprising, for example, administering to the subject an effective amount of nuclease(s) and donor(s) as described herein (e.g., a three-component composition comprising an hIDS transgene and zinc finger nucleases (ZFN)), wherein the subject has reduced, stabilized or eliminated urine GAGs (e.g. urine GAG levels) after treatment. In some embodiments, the delayed or reduced need for a bone marrow transplant is measured, for example, by a change from base line or stabilization in distance walked as measured by a 6-minute walk test.

In some embodiments, the delayed, reduced or prevented need for a bone marrow transplant is measured, for example, by a change from baseline or stabilization in forced vital capacity measured by a pulmonary function test. In some embodiments, the reduced, stabilized or eliminated urine GAGs is measured for the subject after receiving a total dose of between 5e12 vg/kg to 1e15 vg/kg (for example, between 5e12 vg/kg and 5e13 vg/kg, between 5e12 vg/kg and 1e14 vg/kg, between 5e12 vg/kg and 5e14 vg/kg and/or between 5e12 vg/kg and 1e15 vg/kg). In some embodiments, the reduced, stabilized or eliminated urine GAGs is measured for the subject, for example after a treatment with a composition of the invention at a total dose of 5e12 vg/kg, of 1e13 vg/kg, of 5e13 vg/kg, of 1e14 vg/kg, of 5e14 vg/kg and/or 1e15 vg/kg. In some embodiment, provided herein is a method of delaying, reducing or preventing the need for a bone marrow transplant in a subject with MPS II as compared with a subject that has not been treated with the methods and compositions of the invention as disclosed herein, the method comprising administering to the subject an effective amount of hIDS transgene and zinc finger nucleases (ZFN) wherein the subject has a delayed, reduced or prevented need, for porni com example, for a bone marrow transplant after treatment with the methods and compositions disclosed herein.

In some embodiments, provided herein is a method of improving, delaying a decline or maintaining the functional ability in a subject with MPS II by treating the subject with a standard dosing regimen, for example, of ERT in combination with treatment with a composition of the invention as disclosed herein, as compared with a subject that has not been treated, the method comprising administering to the subject an effective amount of hIDS transgene and zinc finger nucleases (ZFN) and with a standard ERT dose, wherein the subject has, for example, an improvement in functional ability, a delay in decline or maintenance of functional ability after treatment. In some embodiments, the treatment using the methods and compositions as disclosed herein of the subject comprises dosing of a composition of the invention, for example, via a peripheral vein catheter. In some embodiments, the methods and compositions disclosed herein comprises dosing of a composition (e.g. via a peripheral vein catheter).

In some embodiments, the hIDS transgene (e.g. SEQ ID NO:15) is delivered (e.g. to the hepatocyte) via AAV2/6 delivery, and the hIDS delivery vector further comprises homology arms (e.g. SEQ ID NO:13 and SEQ ID NO:16) flanking the hIDS transgene with specificity for the regions flanking the ZFN cut site in the albumin locus. In some embodiments, the hIDS transgene (e.g. SEQ ID NO:15) is delivered (e.g. to the hepatocyte) via AAV2/6 delivery, and the hIDS delivery vector further comprises, for example, homology arms (e.g. SEQ ID NO:13 and SEQ ID NO:16) flanking the hIDS transgene with specificity for the regions flanking the ZFN cut site in the albumin locus. In some embodiments, the hIDS transgene (e.g. SEQ ID NO:15) is delivered to the hepatocyte via AAV2/6 delivery, and the hIDS delivery vector further comprises homology arms (e.g. SEQ ID NO:13 and SEQ ID NO:16) flanking the hIDS transgene with specificity for the regions flanking the ZFN cut site in the albumin locus.

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