Process for the preparation of a composition of genetically modified hematopoietic progenitor cells

Abstract

Described are compositions and methods relating to gene therapy, particularly as applied to hematopoietic progenitor (HP) cells, to transduced cells and methods of obtaining them, and to methods of using them to provide prolonged engraftment of modified hematopoietic cells in human subjects. The invention particularly relates to ex vivo gene therapy of HP cells for treatment or prevention of HIV infection.

1 . A composition comprising a pharmaceutically acceptable carrier and at least 1.63×10 6  CD34 +  hematopoietic cells per kg of body weight of a human subject to whom the composition is to be administered, at least 0.52×10 6  of such CD34 +  hematopoietic cells being transduced with a viral construct which expresses an anti-HIV agent. 
     
     
         2 . The composition of  claim 1 , comprising at least 9.37×10 6  CD34 +  hematopoietic cells per kg of body weight of a human subject, wherein at least 5×10 6  of such CD34 +  hematopoietic cells are transduced. 
     
     
         3 . The composition of  claim 1 , wherein the anti-HIV agent is an RNA. 
     
     
         4 . The composition of  claim 1 , wherein the anti-HIV agent is an RNAi molecule. 
     
     
         5 . The composition of  claim 1 , wherein the anti-HIV agent is an antisense molecule. 
     
     
         6 . The composition of  claim 1 , wherein the anti-HIV agent is a ribozyme. 
     
     
         7 . The composition of  claim 1 , wherein the anti-HIV agent is a ribozyme comprising nucleotides having the sequence 5′-UUA GGA UCC UGA UGA GUC CGU GAG GAC GAA ACU GGC UCC-3′. 
     
     
         8 . The composition of  claim 1 , wherein the viral construct is a retroviral construct. 
     
     
         9 . The composition of  claim 1 , wherein the composition is substantially free of cytokines. 
     
     
         10 . The composition of  claim 1 , wherein the composition is substantially free of virus. 
     
     
         11 . The composition of  claim 1 , wherein the transduced CD34 +  cells are capable of engraftment, and of giving rise to progeny cells for at least 12 months, in the subject. 
     
     
         12 . A composition comprising a pharmaceutically acceptable carrier and at least 1.63×10 6  CD34 +  hematopoietic cells per kg of body weight of the subject to whom the composition is to be administered, at least 0.52×10 6  of such CD34 +  hematopoietic cells being transduced with a viral construct which expresses an anti-HIV agent, wherein the composition is produced by a process comprising the steps of:
 (a) isolating CD34 +  hematopoietic cells from the subject; 
 (b) culturing the CD34 +  hematopoietic cells with at least one cytokine; 
 (c) transducing the CD34 +  hematopoietic cells with the viral construct which expresses the anti-HIV agent in the presence of an agent which enhances colocalization of the cells and the viral construct; 
 (d) washing the CD34 +  hematopoietic cells, and 
 (e) mixing the CD34 +  hematopoietic cells with a pharmaceutically acceptable carrier, to thereby obtain the composition. 
 
     
     
         13 . The composition of  claim 12 , wherein the culturing of step (b) is performed in the presence of at least two cytokines. 
     
     
         14 . The composition of  claim 12 , wherein the culturing of step (b) is performed in the presence of two cytokines. 
     
     
         15 . The composition of  claim 12 , wherein the transduction of the cells in step (c) is performed in the presence of a recombinant fibronectin fragment. 
     
     
         16 . A composition comprising a pharmaceutically acceptable carrier and at least 1.63×10 6  CD34 +  hematopoietic cells per kg of body weight of the human subject to whom the composition is to be administered, at least 0.52×10 6  CD34 +  of such CD34 +  hematopoietic cells being transformed with a gene of interest not found in the CD34 +  cells prior to transformation. 
     
     
         17 . The composition of  claim 16 , comprising at least 9×10 6  CD34 +  hematopoietic cells per kg of body weight of a human subject, wherein at least 5×10 6  CD34 +  hematopoietic cells are transduced. 
     
     
         18 . The composition of  claim 16 , wherein the gene of interest expresses an RNA agent. 
     
     
         19 . The composition of  claim 16 , wherein the subject is an adult. 
     
     
         20 - 42 . (canceled)