Post-natal transplantation of factor viii-expressing cells for treatment of hemophilia

Abstract

Disclosed herein are method of treating hemophilia A in a subject comprising injecting the subject with mesenchymal stromal/stem cells (MSC) modified to express high levels of Factor VIII protein. The MSC are isolated prenatally, at birth, or after the subject's birth. The modified MSC may also express high levels von Willebrand factor protein.

1 . A method of treating a subject diagnosed with hemophilia A, comprising:
 (a) modifying mesenchymal stem/stromal cells (MSC) to express high levels of Factor VIII protein or high levels of both Factor VIII protein and von Willebrand factor (vWF) protein thereby generating modified MSC, the MSC comprising bone-marrow MSC isolated from the subject;   (b) generating an expanded modified MSC population by in vitro culturing the modified MSC; and   (c) injecting MSC from the expanded modified MSC population into the subject.   
     
     
         2 . The method of  claim 1 , wherein isolating the MSC comprises isolating cells that express at least one of Stro-1 or CD146. 
     
     
         3 . The method of  claim 1 , wherein the subject has received prior treatment with exogenous Factor VIII and has developed an inhibitory immune response that diminishes the effectiveness of the exogenous Factor VIII treatment. 
     
     
         4 . A method of treating a subject prenatally diagnosed as having hemophilia A, comprising:
 (a) modifying mesenchymal stem/stromal cells (MSC) to express high levels of Factor VIII protein or high levels of both Factor VIII protein and von Willebrand factor (vWF) protein thereby generating modified MSC, the MSC comprising MSC isolated from at least one of amniotic fluid, placental tissue, or umbilical cord tissue obtained at the time of the subject's birth or prenatally from the subject's mother;   (b) generating an expanded modified MSC population by in vitro culturing the modified MSC; and   (c) injecting MSC from the expanded modified MSC population into the subject.   
     
     
         5 . The method of  claim 4 , wherein isolating the MSC comprises isolating cells that express c-kit. 
     
     
         6 . The method of  claim 4 , wherein the isolated MSC express c-kit, CD34, CD90, and CD133. 
     
     
         7 . (canceled) 
     
     
         8 . (canceled) 
     
     
         9 . The method of  claim 1 , wherein the modified MSC comprise one or both of a Factor VIII gene sequence or a vWF gene sequence comprising one or more modifications that increase protein expression, protein stability, or both, of the Factor VIII protein or vWF protein. 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . The method  claim 1 , wherein the MSC are modified by introducing into the MSC
 a viral vector comprising a Factor VIII gene sequence operatively linked to a constitutively active promoter,   a viral vector comprising a vWF gene sequence operatively linked to a constitutively active promoter, or   a viral vector comprising a Factor VIII gene sequence operatively linked to a constitutively active promoter and a vWF gene sequence operatively linked to a constitutively active promoter.   
     
     
         13 . The method  claim 1 , wherein the MSC are modified via gene editing, wherein the gene editing introduces one or more modifications to one or both of an endogenous Factor VIII gene sequence or an endogenous vWF gene sequence that increase protein expression, protein stability, or both. 
     
     
         14 . (canceled) 
     
     
         15 . (canceled) 
     
     
         16 . The method of  claim 12 , wherein the Factor VIII gene sequence and the vWF gene sequence are operatively linked to the same constitutively active promoter. 
     
     
         17 . (canceled) 
     
     
         18 . The method of  claim 1 , wherein the MSC from the expanded modified MSC population are injected into the subject via at least one of intraperitoneal injection, intravenous injection, or intra-articular injection. 
     
     
         19 . The method of  claim 1 , wherein the expanded modified MSC population are injected into the subject at least once, at least twice, or at least three times. 
     
     
         20 . The method of  claim 1 , wherein the expanded modified MSC population are injected into the subject in an amount of about 10 7  to about 10 9  MSC per kilogram weight of the subject. 
     
     
         21 . The method of  claim 4 , wherein the modified MSC comprise one or both of a Factor VIII gene sequence or a vWF gene sequence comprising one or more modifications that increase protein expression, protein stability, or both, of the Factor VIII protein or vWF protein. 
     
     
         22 . The method  claim 4 , wherein the MSC are modified by introducing into the MSC
 a viral vector comprising a Factor VIII gene sequence operatively linked to a constitutively active promoter,   a viral vector comprising a vWF gene sequence operatively linked to a constitutively active promoter, or   a viral vector comprising a Factor VIII gene sequence operatively linked to a constitutively active promoter and a vWF gene sequence operatively linked to a constitutively active promoter.   
     
     
         23 . The method of  claim 22 , wherein the Factor VIII gene sequence and the vWF gene sequence are operatively linked to the same constitutively active promoter. 
     
     
         24 . The method  claim 4 , wherein the MSC are modified via gene editing, wherein the gene editing introduces one or more modifications to one or both of an endogenous Factor VIII gene sequence or an endogenous vWF gene sequence that increase protein expression, protein stability, or both. 
     
     
         25 . The method  claim 4 , wherein the MSC from the expanded modified MSC population are injected into the subject via at least one of intraperitoneal injection, intravenous injection, or intra-articular injection. 
     
     
         26 . The method  claim 4 , wherein the expanded modified MSC population are injected into the subject at least once, at least twice, or at least three times. 
     
     
         27 . The method  claim 4 , wherein the expanded modified MSC population are injected into the subject in an amount of about 10 7  to about 10 9  MSC per kilogram weight of the subject.