Means and methods for nucleic acid delivery vehicle design and nucleic acid transfer

Abstract

Cells capable of at least, in part, complementing adenovirus E2A function of an adenovirus defective in E2A function. Such cells include a nucleic acid encoding adenovirus E2A or a functional part, derivative and/or analogue thereof, integrated into the cell's genome. The cell may have E2A nucleic acid derived from a temperature sensitive adenovirus. Methods for producing an adenovirus particle containing an adenovirus vector with a functional deletion of E2A are also disclosed. Such methods involve providing a cell with the functionally deleted adenovirus vector, culturing the cell, and harvesting viral particle. The functional deletion can comprise a deletion of nucleic acid encoding E2A. In such a method, the nucleic acid encoding adenovirus E2A in the cell's genome has no sequence overlap with the vector leading to replication competent adenovirus and/or to the formation of an adenovirus vector comprising E2A function. In the method, the adenovirus vector may further include a functional deletion of E1-region encoding nucleic acid. Methods for providing cells of an individual with a nucleic acid of interest, without risk of administering simultaneously a replication competent adenovirus vector, comprising administering the individual one of the previously described preparations are also disclosed.

What is claimed is:  
     
         1 . A cell capable of at least in part complementing adenovirus E2A function of an adenovirus defective in E2A function, said cell comprising a nucleic acid encoding adenovirus E2A or a functional part thereof, derivative thereof, analogue thereof, or mixture of any of these.  
     
     
         2 . The cell of  claim 1  wherein said nucleic acid encoding adenovirus E2A is a temperature sensitive adenovirus.  
     
     
         3 . The cell of  claim 2  wherein said nucleic acid encoding adenovirus E2A is of adenovirus ts125 origin.  
     
     
         4 . The cell of  claim 1 ,  claim 2 , or  claim 3  further comprising a nucleic acid encoding adenovirus E1-region proteins or a functional part, derivative thereof, analogue thereof, or mixture of any thereof.  
     
     
         5 . The cell of  claim 4  wherein said cell is of PER.C6 (ECACC deposit number 96022940) origin.  
     
     
         6 . A method for producing an adenoviral particle containing an adenovirus vector with a functional deletion of E2A, said method comprising: 
 providing a cell according to  claim 1 ,  claim 2 ,  claim 3 ,  claim 4 , or  claim 5  with said    adenovirus vector,    culturing said cell, and    harvesting said adenoviral particle.    
     
     
         7 . The method for producing an adenoviral particle containing an adenovirus vector with a functional deletion of E2A according to  claim 6  wherein said functional deletion comprises a deletion of at least part of the nucleic acid encoding E2A.  
     
     
         8 . The method for producing an adenoviral particle containing an adenovirus vector with a functional deletion of E2A according to  claim 6  wherein said nucleic acid encoding adenovirus E2A in said cell's genome does not comprise sequence overlap with said vector which leads to replication competent adenovirus and/or to the formation of an adenovirus vector comprising E2A function.  
     
     
         9 . The method for producing an adenoviral particle according to  claim 6 , said method further comprising: 
 providing an adenovirus vector further comprising a functional deletion of E1-region encoding nucleic acid said adenovirus vector to said cell, said cell further characterized in: 
 being capable of at least in part complementing adenovirus E2A function of an adenovirus defective in E2A function,  
 comprising a nucleic acid encoding adenovirus E2A or a functional part thereof, derivative thereof, and/or analogue thereof, and  
 further comprising a nucleic acid sequence encoding adenovirus E1-region proteins or a functional part, derivative thereof, and/or analogue thereof,  
   culturing said cell, and    harvesting said virus particle.    
     
     
         10 . The method according to  claim 9  wherein said nucleic acid encoding adenovirus E1-region has no sequence overlap with said vector which leads to replication competent adenovirus and/or to the formation of an adenovirus vector comprising an E1 function.  
     
     
         11 . A method according to  claim 6 ,  claim 7 ,  claim 8 ,  claim 9 , or  claim 10  wherein said adenovirus vector further comprises at least one nucleic acid of interest.  
     
     
         12 . An adenovirus vector comprising a functional deletion of adenovirus E2A.  
     
     
         13 . The adenovirus vector of  claim 12  wherein said vector comprises a deletion of at least part of the nucleic acid encoding E2A.  
     
     
         14 . The adenovirus vector of  claim 13  wherein said deletion encompasses at least the entire coding region of E2A.  
     
     
         15 . The adenovirus vector of  claim 14  further comprising a deletion corresponding to a deletion of nucleotides 22443 to 24032 in adenovirus 5.  
     
     
         16 . The adenovirus vector of  claim 12 ,  claim 13 ,  claim 14 , or  claim 15  further comprising a deletion of nucleic acid encoding E1-region proteins or parts, derivatives and/or analogues thereof.  
     
     
         17 . The adenovirus vector of  claim 12 ,  claim 13 ,  claim 14 ,  claim 15 , or  claim 16  wherein said deletion of nucleic acid encoding E1-region proteins comprises a deletion corresponding to a deletion of nucleotides 459 to 3510 in adenovirus 5.  
     
     
         18 . The adenovirus vector of  claim 12 ,  claim 13 ,  claim 14 ,  claim 15 ,  claim 16 , or  claim 17  further comprising at least one nucleic acid of interest.  
     
     
         19 . A preparation of adenovirus vector containing adenovirus particles wherein said adenovirus vector comprises a functional deletion of E2A.  
     
     
         20 . The preparation of  claim 19  wherein said adenovirus vector further comprises a deletion of nucleic acid encoding E1-region proteins or parts, derivatives and/or analogues thereof.  
     
     
         21 . The preparation of  claim 19  or  claim 20  free of adenovirus vectors comprising E2A function.  
     
     
         22 . The preparation of  claim 21 , said preparation characterized in being free of adenovirus vectors comprising nucleic acid encoding a functional E2A, or a functional part, derivative and/or analogue thereof.  
     
     
         23 . The preparation according to  claim 21  or  claim 22  free of adenovirus vectors comprising nucleic acid encoding E1-region proteins or parts, derivatives and/or analogues thereof.  
     
     
         24 . A method for providing cells of an individual with a nucleic acid of interest, without risk of administering simultaneously a replication competent adenovirus vector, comprising administering said individual a preparation according to  claim 19 ,  claim 20 ,  claim 21 ,  claim 22 , or  claim 23 .  
     
     
         25 . The method of  claim 24  wherein said preparation is characterized in being free of adenovirus vectors comprising nucleic acid encoding a functional E2A, or a functional part, derivative and/or analogue thereof.  
     
     
         26 . The method of  claim 24  or  claim 25  wherein said preparation is free of adenovirus vectors comprising nucleic acid encoding E1-region proteins or parts, derivatives and/or analogues thereof.  
     
     
         27 . An adenovirus vector comprising at least a deletion of a region which in adenovirus 5 corresponds to nucleotides 22443-24032.  
     
     
         28 . An adenovirus vector comprising at least a deletion of a region which in adenovirus 5 corresponds to nucleotides 22418-24037.  
     
     
         29 . An adenovirus vector comprising at least a deletion of a region which in adenovirus 5 corresponds to nucleotides 22348-24060.  
     
     
         30 . An adenovirus vector according to  claim 27 ,  claim 28 , or  claim 29  further comprising at leastnucleic acidwhichin adenovirus 5 corresponds to nucleotides 3534-22347 and/ornucleotides 24061 until the right ITR.  
     
     
         31 . An adenovirus vector according to  claim 27  or  claim 28  further comprising at least nucleic acid which in adenovirus 5 corresponds to nucleotides 3534-22417 and/ornucleotides 24038 until the right ITR.  
     
     
         32 . An adenovirus vector according to  claim 27  further comprising at least nucleic acid which in adenovirus 5 corresponds to nucleotides 3534-22442 and/or nucleotides 24033 until the right ITR.  
     
     
         33 . An adenovirus vector according to  claim 27  further comprising at least nucleic acid which in adenovirus 5 corresponds to nucleotides 3534-22442 and/or nucleotides 24033 until the right ITR.  
     
     
         34 . The cell of  claim 1  wherein said nucleic acid is integrated into said cell's genome.  
     
     
         35 . The cell of  claim 4  wherein said cell is derived from cell line 293.  
     
     
         36 . The method according to  claim 9  wherein said nucleic acid is integrated into said cell's genome.  
     
     
         37 . A cell capable of at least in part complementing adenovirus E2A function of an adenovirus defective in E2A function, said cell comprising a nucleic acid encoding adenovirus E2A or a functional part thereof.  
     
     
         38 . The cell of  claim 37 , said cell further comprising a nucleic acid encoding adenovirus E1-region proteins or a functional part thereof.  
     
     
         39 . The cell of  claim 38 , wherein said nucleic acid encoding adenovirus E2A encodes a temperature sensitive E2A mutant.  
     
     
         40 . The cell of  claim 39 , wherein said temperature sensitive E2A mutant is an E2A mutant encoded by adenovirus ts125.  
     
     
         41 . The cell of  claim 38 , wherein said cell is of cell line 293 origin.  
     
     
         42 . The cell of  claim 39 , wherein said cell is of cell line 293 origin.  
     
     
         43 . The cell of  claim 40 , wherein said cell is of cell line 293 origin.  
     
     
         44 . A method for producing an adenoviral particle containing an adenovirus vector with a functional deletion of E2A, said method comprising: 
 providing a cell according to any one of claims  37 - 43  with said adenovirus vector,    culturing said cell, and    harvesting said adenoviral particle.    
     
     
         45 . The method according to  claim 44 , wherein said functional deletion comprises a deletion of at least part of the nucleic acid encoding E2A.  
     
     
         46 . The method according to  claim 45 , wherein said nucleic acid encoding adenovirus E2A in said cell's genome does not comprise sequence overlap with said vector which leads to replication competent adenovirus and/or to the formation of an adenovirus vector comprising E2A function.  
     
     
         47 . The method according to  claim 45 , said method further comprising: 
 providing an adenovirus vector further comprising a functional deletion of E1-region encoding nucleic acid said adenovirus vector to said cell, said cell further characterized by: 
 being capable of at least in part complementing adenovirus E2A function of an adenovirus defective in E2A function,  
 comprising a nucleic acid encoding adenovirus E2A or a functional part thereof, derivative thereof, and/or analogue thereof, and  
 further comprising a nucleic acid sequence encoding adenovirus E1-region proteins or a functional part, derivative thereof, and/or analogue thereof,  
   culturing said cell, and    harvesting said virus particle.    
     
     
         48 . An adenovirus vector comprising a functional deletion of adenovirus E2A.  
     
     
         49 . The adenovirus vector of  claim 48  wherein said vector comprises a deletion of at least part of the nucleic acid encoding E2A.  
     
     
         50 . The adenovirus vector of  claim 49  wherein said deletion encompasses at least the entire coding region of E2A.  
     
     
         51 . The adenovirus vector of  claim 48 , further having a deletion of nucleic acid encoding E1-region proteins or parts, derivatives and/or analogues thereof.  
     
     
         52 . The adenovirus vector of  claim 49 , further having a deletion of nucleic acid encoding E1-region proteins or parts, derivatives and/or analogues thereof.  
     
     
         53 . The adenovirus vector of  claim 50  further having a deletion of nucleic acid encoding E1-region proteins or parts, derivatives and/or analogues thereof.  
     
     
         54 . A preparation of adenovirus vector containing adenovirus particles wherein said adenovirus vector has a functional deletion of E2A.  
     
     
         55 . The preparation of  claim 54  wherein said adenovirus vector further has a deletion of nucleic acid encoding E1-region proteins or parts, derivatives and/or analogues thereof.  
     
     
         56 . The preparation of  claim 54  free of adenovirus vectors comprising E2A function.  
     
     
         57 . The preparation of  claim 55  free of adenovirus vectors comprising E2A function.  
     
     
         58 . The preparation of  claim 56 , said preparation characterized in being free of adenovirus vectors comprising nucleic acid encoding a functional E2A, or a functional part, derivative and/or analogue thereof.  
     
     
         59 . The preparation of  claim 57 , said preparation characterized in being free of adenovirus vectors comprising nucleic acid encoding a functional E2A, or a functional part, derivative and/or analogue thereof.  
     
     
         60 . The preparation of  claim 56 , free of adenovirus vectors comprising nucleic acid encoding E1-region proteins or parts, derivatives and/or analogues thereof.  
     
     
         61 . The preparation of  claim 57 , free of adenovirus vectors comprising nucleic acid encoding E1-region proteins or parts, derivatives and/or analogues thereof.  
     
     
         62 . The preparation of  claim 58 , free of adenovirus vectors comprising nucleic acid encoding E1-region proteins or parts, derivatives and/or analogues thereof.  
     
     
         63 . The preparation of  claim 59  free of adenovirus vectors comprising nucleic acid encoding E1-region proteins or parts, derivatives and/or analogues thereof.  
     
     
         64 . A method for providing cells of a subject with a nucleic acid of interest, with a decreased risk of administering simultaneously a replication competent adenovirus vector, said method comprising administering to the subject's cells the preparation of any one of claims  54 - 63 .  
     
     
         65 . An adenovirus vector comprising at least a deletion of a region which in adenovirus 5 corresponds to nucleotides 22443-24032, or nucleotides 22418-24037, or nucleotides 22348-24060, further comprising at least nucleic acid which in adenovirus 5 corresponds to nucleotides 3534-22347 and/or nucleotides 24061 until the right ITR.  
     
     
         66 . The method according to  claim 44 , wherein said nucleic acid is integrated into said cell's genome.  
     
     
         67 . The method according to  claim 45 , wherein said nucleic acid is integrated into said cell's genome.  
     
     
         68 . The method according to of  claim 46 , wherein said nucleic acid is integrated into said cell's genome.  
     
     
         69 . The method according to  claim 47 , wherein said nucleic acid is integrated into said cell's genome.