Lyophilizable and enhanced compacted nucleic acids

Abstract

Counterions of polycations used to compact nucleic acids profoundly affect shape and stability of particles formed. Shape is associated with differential serum nuclease resistance and colloidal stability. A surrogate for determining such properties that is easy to measure is the turbidity parameter. Shape also affects the suitability and efficacy of compacted nucleic acid complexes for transfecting cells by various routes into a mammalian body. Moreover, counterions such as acetate can protect compacted nucleic acid complexes from adverse effects of lyophilization

1 . A method of estimating the colloidal stability of a preparation of compacted nucleic acids, comprising the steps of: 
 determining a turbidity parameter of a solution of compacted nucleic acid, wherein the turbidity parameter is defined as the slope of a straight line obtained by plotting log of apparent absorbance of light versus log of incident wavelength of the light, wherein said wavelength is between about 330 nm and 420 nm;    identifying the preparation as colloidally stable if a turbidity parameter of less than −3 is determined and identifying the preparation as colloidally unstable if a turbidity parameter of greater than or equal to −3 is determined.    
     
     
         2 . A non-naturally occurring composition comprising unaggregated nucleic acid complexes, each complex consisting essentially of a single nucleic acid molecule and one or more polycation molecules, said polycation molecules having a counterion selected from the group consisting of acetate, bicarbonate, and chloride, wherein said complex is compacted to a diameter which is less than (a) double the theoretical diameter of a complex of said single nucleic acid molecule and a sufficient number of polycation molecules to provide a charge ratio of about 1:1, in the form of a condensed sphere, or (b) 30 nm, whichever is larger.  
     
     
         3 . The composition of  claim 2  wherein the polycation molecules are polylysine or a polylysine derivative.  
     
     
         4 . The composition of  claim 3  wherein the polylysine derivative is polylysine peptide with a cysteine residue.  
     
     
         5 . The composition of  claim 2 , said complex is compacted to a diameter of less than 90 nm.  
     
     
         6 . The composition of  claim 2 , wherein the nucleic acid complex is compacted to a diameter less than 30 nm.  
     
     
         7 . The composition of  claim 2 , wherein the nucleic acid complex is compacted to a diameter less than 23 nm.  
     
     
         8 . The composition of  claim 2 , wherein the nucleic acid complex is compacted to a diameter not more than 12 nm.  
     
     
         9 . The composition of  claim 2  wherein said complex is compacted to a diameter which is less than double the theoretical diameter of a complex of said single nucleic acid and a sufficient number of positively charged residues to provide a charge ratio of about 1:1, in the form of a condensed sphere.  
     
     
         10 . A method of preparing a composition according to  claim 2  which comprises mixing the nucleic acid with the polycation having acetate as a counterion, at a salt concentration sufficient for compaction of the complex.  
     
     
         11 . The method of  claim 10  in which the mixing is monitored to detect, prevent or correct, the formation of aggregated or relaxed complexes.  
     
     
         12 . The method of  claim 10  wherein the salt is NaCl.  
     
     
         13 . The method of  claim 10  wherein the nucleic acid and the polycation are each, at the time of mixing, in a solution having a salt concentration of 0.05 to 1.5 M.  
     
     
         14 . The method of  claim 10  in which the molar ratio of the phosphate groups of the nucleic acid to the positively charged groups of the polycation is in the range of 4:1 to 1:4.  
     
     
         15 . The method of  claim 10  in which the polycation is added to the nucleic acid, while vortexing at high speed.  
     
     
         16 . The method of  claim 10  in which the nucleic acid is added to the polycation, while vortexing at high speed.  
     
     
         17 . The method of  claim 10  wherein the mixing is monitored by a method selected from the group consisting of electron microscopy, light scattering, circular dichroism, and absorbance measurement.  
     
     
         18 . The method of  claim 10  wherein the polycation molecules are polylysine or a polylysine derivative.  
     
     
         19 . The method of  claim 18  wherein the polylysine derivative is polylysine peptide with a cysteine residue.  
     
     
         20 . A non-naturally occurring composition comprising unaggregated nucleic acid complexes, each complex consisting essentially of a single nucleic acid molecule and one or more polycation molecules, wherein said polycation molecules have a counterion selected from the group consisting of acetate, bicarbonate, and chloride, said polycation molecule having a nucleic acid binding moiety through which it is complexed to the nucleic acid, wherein said nucleic acid molecule encodes at least one functional protein, wherein said complex is compacted to a diameter which is less than double the theoretical minimum diameter of a complex of said single nucleic acid molecule and a sufficient number of polycation molecules to provide a charge ratio of about 1:1, in the form of a condensed sphere, or 30 nm, whichever is larger.  
     
     
         21 . The composition of  claim 20  wherein the polycation molecules are polylysine or a polylysine derivative.  
     
     
         22 . The composition of  claim 21  wherein the polylysine derivative is polylysine peptide with a cysteine residue.  
     
     
         23 . The non-naturally occurring composition of  claim 20  wherein said nucleic acid molecule comprises a promoter which controls transcription of an RNA molecule encoding the functional protein.  
     
     
         24 . The non-naturally occurring composition of  claim 20  wherein the protein is therapeutic.  
     
     
         25 . The non-naturally occurring composition of  claim 20  wherein the complexe is compacted to a diameter which is less than 50 nm.  
     
     
         26 . The non-naturally occurring composition of  claim 20  wherein the complex is compacted to a diameter which is less than 30 nm.  
     
     
         27 . The non-naturally occurring composition of  claim 20  wherein the nucleic acid complex is compacted to a diameter less than 23 nm.  
     
     
         28 . The non-naturally occurring composition of  claim 20  wherein the nucleic acid complex is compacted to a diameter not more than 12 nm.  
     
     
         29 . A non-naturally occurring composition comprising unaggregated nucleic acid complexes, each complex consisting essentially of a single double-stranded cDNA molecule and one or more polycation molecules, said polycation molecules having a counterion selected from the group consisting of acetate, bicarbonate, and chloride, wherein said cDNA molecule encodes at least one functional protein, wherein said complex is compacted to a diameter which is less than double the theoretical minimum diameter of a complex of said single cDNA molecule and a sufficient number of polycation molecules to provide a charge ratio of about 1:1, in the form of a condensed sphere, or 30 nm, whichever is larger.  
     
     
         30 . The composition of  claim 29  wherein the polycation molecules are polylysine or a polylysine derivative.  
     
     
         31 . The composition of  claim 30  wherein the polylysine derivative is polylysine peptide with a cysteine residue.  
     
     
         32 . A non-naturally occurring composition comprising unaggregated nucleic acid complexes, each complex consisting essentially of a single nucleic acid molecule and one or more polycation molecules, said polycation molecules having a counterion selected from the group consisting of acetate, bicarbonate, and chloride, wherein said nucleic acid molecule encodes at least one antisense nucleic acid, wherein said complex is compacted to a diameter which is less than double the theoretical minimum diameter of a complex of said single nucleic acid molecule and a sufficient number of polycation molecules to provide a charge ratio of about 1:1, in the form of a condensed sphere, or 30 nm, whichever is larger.  
     
     
         33 . The composition of  claim 32  wherein the polycation molecules are polylysine or a polylysine derivative.  
     
     
         34 . The composition of  claim 33  wherein the polylysine derivative is polylysine peptide with a cysteine residue.  
     
     
         35 . A non-naturally occurring composition comprising unaggregated nucleic acid complexes, each complex consisting essentially of a single nucleic acid molecule and one or more polycation molecules, said polycation molecule having a counterion selected from the group consisting of acetate, bicarbonate, and chloride, wherein said nucleic acid molecule is an RNA molecule, wherein said complex is compacted to a diameter which is less than double the theoretical minimum diameter of a complex of said single nucleic acid molecule and a sufficient number of polycation molecules to provide a charge ratio of about 1:1, in the form of a condensed sphere, or 30 nm, whichever is larger.  
     
     
         36 . The composition of  claim 35  wherein the polycation molecules are polylysine or a polylysine derivative.  
     
     
         37 . The composition of  claim 36  wherein the polylysine derivative is polylysine peptide with a cysteine residue.  
     
     
         38 . A method of preparing a composition comprising unaggregated nucleic acid complexes, each complex consisting essentially of a single nucleic acid molecule and one or more polycation molecules, said method comprising: 
 mixing a nucleic acid molecule with a polycation molecule at a salt concentration sufficient for compaction of the complex to a diameter which is less than double the theoretical minimum diameter of a complex of said single nucleic acid molecule and a sufficient number of polycation molecules to provide a charge ratio of about 1:1, in the form of a condensed sphere, or 30 nm, whichever is larger, whereby unaggregated nucleic acid complexes are formed, wherein each complex consists essentially of a single nucleic acid molecule and one or more polycation molecules, and wherein said polycation molecules have a counterion selected from the group consisting of bicarbonate and chloride.    
     
     
         39 . The method of  claim 38  wherein the polycation molecules are polylysine or a polylysine derivative.  
     
     
         40 . The method of  claim 39  wherein the polylysine derivative is polylysine peptide with a cysteine residue.  
     
     
         41 . A method of preparing a composition comprising unaggregated nucleic acid complexes, each complex consisting essentially of a single nucleic acid molecule and one or more polycation molecules, said method comprising: 
 mixing a nucleic acid molecule with a polycation molecule in a solvent to form a complex, said mixing being performed in the absence of added salt, whereby the nucleic acid forms soluble complexes with the polycation molecule without forming aggregates, wherein each complex consists essentially of a single nucleic acid molecule and one or more polycation molecules, wherein the complexes have a diameter which is less than double the theoretical minimum diameter of a complex of said single nucleic acid molecule and a sufficient number of polycation molecules to provide a charge ratio of about 1:1, in the form of a condensed sphere, or 30 nm, whichever is larger, wherein the polycation has acetate as a counterion.    
     
     
         42 . The method of  claim 41  wherein the polycation molecules are polylysine or a polylysine derivative.  
     
     
         43 . The method of  claim 42  wherein the polylysine derivative is polylysine peptide with a cysteine residue.  
     
     
         44 . A method of preparing a composition comprising unaggregated nucleic acid complexes, each complex consisting essentially of a single nucleic acid molecule and one or more polycation molecules, said method comprising: 
 mixing a nucleic acid molecule with a polycation molecule in a solvent to form a complex, said mixing being performed in the absence of added salt, whereby the nucleic acid forms soluble complexes with the polycation molecule without forming aggregates, wherein each complex consists essentially of a single nucleic acid molecule and one or more polycation molecules, wherein the complexes have a diameter which is less than double the theoretical minimum diameter of a complex of said single nucleic acid molecule and a sufficient number of polycation molecules to provide a charge ratio of about 1:1, in the form of a condensed sphere, or 30 nm, whichever is larger, wherein the polycation has a counterion selected from the group consisting of bicarbonate and chloride.    
     
     
         45 . The method of  claim 44  wherein the polycation molecules are polylysine or a polylysine derivative.  
     
     
         46 . The method of  claim 45  wherein the polylysine derivative is polylysine peptide with a cysteine residue.  
     
     
         47 . Non-naturally occurring, soluble compacted complexes of a nucleic acid and a polycation molecule made by the process of  claim 10 .  
     
     
         48 . Non-naturally occurring, soluble compacted complexes of a nucleic acid and a polycation molecule made by the process of  claim 38 .  
     
     
         49 . Non-naturally occurring, soluble compacted complexes of a nucleic acid and a polycation molecule made by the process of  claim 41 .  
     
     
         50 . Non-naturally occurring, soluble compacted complexes of a nucleic acid and a polycation made by the process of  claim 44 .  
     
     
         51 . The complexes of  claim 47  wherein the polycation molecules are polylysine or a polylysine derivative.  
     
     
         52 . The complexes of  claim 51  wherein the polylysine derivative is polylysine peptide with a cysteine residue  
     
     
         53 . The complexes of  claim 48  wherein the polycation molecules are polylysine or a polylysine derivative.  
     
     
         54 . The complexes of  claim 53  wherein the polylysine derivative is polylysine peptide with a cysteine residue.  
     
     
         55 . The complexes of  claim 49  wherein the polycation molecules are polylysine or a polylysine derivative.  
     
     
         56 . The complexes of  claim 55  wherein the polylysine derivative is polylysine peptide with a cysteine residue.  
     
     
         57 . The complexes of  claim 50  wherein the polycation molecules are polylysine or a polylysine derivative.  
     
     
         58 . The complexes of  claim 57  wherein the polylysine derivative is polylysine peptide with a cysteine residue.  
     
     
         59 . A method of preventing or treating a disease or other clinical condition in a subject which comprises: 
 administering intramuscularly or to the lung of the subject a prophylactically or therapeutically effective amount of a composition comprising: 
 unaggregated nucleic acid complexes, each complex consisting essentially of a single nucleic acid molecule and one or more polycation molecules, said polycation molecule having acetate as a counterion, wherein said complex is compacted to a diameter which is less than (a) double the theoretical minimum diameter of a complex of said single nucleic acid molecule and a sufficient number of polycation molecules to provide a charge ratio of about 1:1, in the form of a condensed sphere, or (b) 30 nm, whichever is larger,  
   said nucleic acid being one whose integration, hybridization or expression within target cells of said subject prevents or treats said disease or other clinical condition.    
     
     
         60 . The method of  claim 59  wherein the step of administering is by inhalation.  
     
     
         61 . The method of  claim 59  wherein the step of administering is by intramuscular injection.  
     
     
         62 . The method of  claim 59  wherein the polycation molecules are polylysine or a polylysine derivative.  
     
     
         63 . The method of  claim 62  wherein the polylysine derivative is polylysine peptide with a cysteine residue.  
     
     
         64 . A method of preventing or treating a disease or other clinical condition in a subject which comprises: 
 administering intramuscularly or to the lung of the subject a prophylactically or therapeutically effective amount of a composition comprising: 
 unaggregated nucleic acid complexes, each complex consisting essentially of a single nucleic acid molecule and one or more polycation molecules, said polycation molecule having a counterion selected from the group consisting of bicarbonate and chloride,  
 wherein said complex is compacted to a diameter which is less than (a) double the theoretical minimum diameter of a complex of said single nucleic acid molecule and a sufficient number of polycation molecules to provide a charge ratio of about 1:1, in the form of a condensed sphere, or (b) 30 nm, whichever is larger,  
 said nucleic acid being one whose integration, hybridization or expression within target cells of said subject prevents or treats said disease or other clinical condition.  
   
     
     
         65 . The method of  claim 64  wherein the polycation molecules are polylysine or a polylysine derivative.  
     
     
         66 . The method of  claim 65  wherein the polylysine derivative is polylysine peptide with a cysteine residue.  
     
     
         67 . The method of  claim 64  wherein the step of administering is by inhalation.  
     
     
         68 . The method of  claim 64  wherein the step of administering is by intramuscular injection.  
     
     
         69 . The composition of  claim 20  wherein said complex is compacted to a diameter which is less than double the theoretical diameter of a complex of said single nucleic acid and a sufficient number of positively charged residues to provide a charge ratio of about 1:1, in the form of a condensed sphere.  
     
     
         70 . The composition of  claim 29  wherein the nucleic acid complexes are associated with a lipid.  
     
     
         71 . The composition of  claim 29  wherein said complex is compacted to a diameter of less than 90 nm.  
     
     
         72 . The composition of  claim 29  wherein the nucleic acid complex is compacted to a diameter less than 30 nm.  
     
     
         73 . The composition of  claim 29  wherein the nucleic acid complex is compacted to a diameter less than 23 nm.  
     
     
         74 . The composition of  claim 29  wherein the nucleic acid complex is compacted to a diameter not more than 12 nm.  
     
     
         75 . The composition of  claim 29  wherein said complex is compacted to a diameter which is less than double the theoretical diameter of a complex of said single nucleic acid and a sufficient number of positively charged residues to provide a charge ratio of about 1:1, in the form of a condensed sphere.  
     
     
         76 . The composition of  claim 32  wherein said complex is compacted to a diameter of less than 90 nm.  
     
     
         77 . The composition of  claim 32  wherein the nucleic acid complex is compacted to a diameter less than 30 nm.  
     
     
         78 . The composition of  claim 32  wherein the nucleic acid complex is compacted to a diameter less than 23 nm.  
     
     
         79 . The composition of  claim 32  wherein the nucleic acid complex is compacted to a diameter not more than 12 nm.  
     
     
         80 . The composition of  claim 32  wherein said complex is compacted to a diameter which is less than double the theoretical diameter of a complex of said single nucleic acid and a sufficient number of positively charged residues to provide a charge ratio of about 1:1, in the form of a condensed sphere.  
     
     
         81 . The composition of  claim 35  said complex is compacted to a diameter of less than 90 nm.  
     
     
         82 . The composition of  claim 35  wherein the nucleic acid complex is compacted to a diameter less than 30 nm.  
     
     
         83 . The composition of  claim 35  wherein the nucleic acid complex is compacted to a diameter less than 23 nm.  
     
     
         84 . The composition of  claim 35  wherein the nucleic acid complex is compacted to a diameter not more than 12 nm.  
     
     
         85 . The composition of  claim 35  wherein said complex is compacted to a diameter which is less than double the theoretical diameter of a complex of said single nucleic acid and a sufficient number of positively charged residues to provide a charge ratio of about 1:1, in the form of a condensed sphere.  
     
     
         86 . The method of  claim 38  wherein the salt is NaCl.  
     
     
         87 . The method of  claim 38  wherein the nucleic acid and the polycation are each, at the time of mixing, in a solution having a salt concentration of 0.05 to 1.5 M.  
     
     
         88 . The method of  claim 38  in which the mixing is monitored to detect, prevent or correct, the formation of aggregated or relaxed complexes.  
     
     
         89 . The method of  claim 38  in which the molar ratio of the phosphate groups of the nucleic acid to the positively charged groups of the polycation is in the range of 4:1 to 1:4.  
     
     
         90 . The method of  claim 38  in which the polycation is added to the nucleic acid, while vortexing at high speed.  
     
     
         91 . The method of  claim 38  in which the nucleic acid is added to the polycation, while vortexing at high speed.  
     
     
         92 . The method of  claim 38  wherein the mixing is monitored by a method selected from the group consisting of electron microscopy, light scattering, circular dichroism, and absorbance measurement.  
     
     
         93 . The method of  claim 41  in which the mixing is monitored to detect, prevent or correct, the formation of aggregated or relaxed complexes.  
     
     
         94 . The method of  claim 41  in which the molar ratio of the phosphate groups of the nucleic acid to the positively charged groups of the polycation is in the range of 4:1 to 1:4.  
     
     
         95 . The method of  claim 41  in which the polycation is added to the nucleic acid, while vortexing at high speed.  
     
     
         96 . The method of  claim 41  in which the nucleic acid is added to the polycation, while vortexing at high speed.  
     
     
         97 . The method of  claim 41  wherein the mixing is monitored by a method selected from the group consisting of electron microscopy, light scattering, circular dichroism, and absorbance measurement.  
     
     
         98 . The method of  claim 44  in which the mixing is monitored to detect, prevent or correct, the formation of aggregated or relaxed complexes.  
     
     
         99 . The method of  claim 44  in which the molar ratio of the phosphate groups of the nucleic acid to the positively charged groups of the polycation is in the range of 4:1 to 1:4.  
     
     
         100 . The method of  claim 44  in which the polycation is added to the nucleic acid, while vortexing at high speed.  
     
     
         101 . The method of  claim 44  in which the nucleic acid is added to the polycation, while vortexing at high speed.  
     
     
         102 . The method of  claim 44  wherein the mixing is monitored by a method selected from the group consisting of electron microscopy, light scattering, circular dichroism, and absorbance measurement.  
     
     
         103 . A non-naturally occurring composition comprising unaggregated nucleic acid complexes, each complex consisting essentially of a single nucleic acid molecule and one or more polycation molecules, said polycation molecules having a counterion selected from the group consisting of acetate, bicarbonate, and chloride.  
     
     
         104 . The composition of  claim 103  wherein the counterion is acetate.  
     
     
         105 . The composition of  claim 2  wherein said polycation is CK15-60P10 and the counterion is acetate, wherein CK15-60P10 is a polyamino acid polymer of one N-terminal cysteine and 15-60 lysine residues, wherein a molecule of polyethylene glycol having an average molecular weight of 10 kdal is attached to the cysteine residue.  
     
     
         106 . The composition of  claim 105  wherein the polycation molecule comprises 30 residues of lysine.  
     
     
         107 . The composition of  claim 105  wherein the polycation molecule comprises a targeting moiety.  
     
     
         108 . The composition of  claim 105 , said complex is compacted to a diameter of less than 90 nm.  
     
     
         109 . The composition of  claim 105 , wherein the nucleic acid complex is compacted to a diameter less than 30 nm.  
     
     
         110 . The composition of  claim 105 , wherein the nucleic acid complex is compacted to a diameter less than 23 nm.  
     
     
         111 . The composition of  claim 105 , wherein the nucleic acid complex is compacted to a diameter not more than 12 nm.  
     
     
         112 . The composition of  claim 105  wherein said complex is compacted to a diameter which is less than double the theoretical diameter of a complex of said single nucleic acid and a sufficient number of positively charged residues to provide a charge ratio of about 1:1, in the form of a condensed sphere.  
     
     
         113 . The composition of  claim 105  which is lyophilized.  
     
     
         114 . The composition of  claim 105  which is rehydrated after lyophilization.  
     
     
         115 . The composition of  claim 105  which does not contain a disaccharide.  
     
     
         116 . A method of delivering polynucleotides to cells comprising: 
 contacting the composition of  claim 114  with cells, whereby the nucleic acid is delivered to and taken up by the cells.    
     
     
         117 . The method of  claim 116  wherein the composition does not contain a disaccharide.  
     
     
         118 . The composition of  claim 20  wherein the polycation is CK15-60P10, and the counterion is acetate, wherein CK15-60 is a polyamino acid polymer of one N-terminal cysteine and 15-60 lysine residues, wherein a molecule of polyethylene glycol having an average molecular weight of 10 kdal is attached to the cysteine residue.  
     
     
         119 . The composition of  claim 118  wherein the polycation molecule comprises 30 residues of lysine.  
     
     
         120 . The composition of  claim 118  wherein the polycation molecule comprises a targeting moiety.  
     
     
         121 . The composition of  claim 118  which is lyophilized.  
     
     
         122 . The non-naturally occurring composition of  claim 118  wherein said nucleic acid molecule comprises a promoter which controls transcription of an RNA molecule encoding the functional protein.  
     
     
         123 . The non-naturally occurring composition of  claim 118  wherein the protein is therapeutic.  
     
     
         124 . The non-naturally occurring composition of  claim 118  wherein the complex is compacted to a diameter which is less than 50 nm.  
     
     
         125 . The non-naturally occurring composition of  claim 118  wherein the complex is compacted to a diameter which is less than 30 nm.  
     
     
         126 . The non-naturally occurring composition of  claim 118  wherein the nucleic acid complex is compacted to a diameter less than 23 nm.  
     
     
         127 . The non-naturally occurring composition of  claim 118  wherein the nucleic acid complex is compacted to a diameter not more than 12 nm.  
     
     
         128 . The composition of  claim 118  wherein said complex is compacted to a diameter which is less than double the theoretical diameter of a complex of said single nucleic acid and a sufficient number of positively charged residues to provide a charge ratio of about 1:1, in the form of a condensed sphere.  
     
     
         129 . The composition of  claim 118  which is rehydrated after lyophilization.  
     
     
         130 . The composition of  claim 118  which does not contain a disaccharide.  
     
     
         131 . A method of delivering polynucleotides to cells comprising: 
 contacting the composition of  claim 129  with cells, wherein the polynucleotide encodes a protein, whereby the protein is expressed.    
     
     
         132 . The composition of  claim 29  wherein said polycation is CK15-60P10, and said counterion is acetate, wherein CK15-60P10 is a polyamino acid polymer of one N-terminal cysteine and 15-60 lysine residues, wherein a molecule of polyethylene glycol having an average molecular weight of 10 kdal is attached to the cysteine residue.  
     
     
         133 . The composition of  claim 132  wherein the polycation molecule comprises 30 residues of lysine.  
     
     
         134 . The composition of  claim 132  wherein the polycation molecule comprises a targeting moiety.  
     
     
         135 . The composition of  claim 132  which is lyophilized.  
     
     
         136 . The composition of  claim 132  wherein said complex is compacted to a diameter which is less than double the theoretical diameter of a complex of said single nucleic acid and a sufficient number of positively charged residues to provide a charge ratio of about 1:1, in the form of a condensed sphere.  
     
     
         137 . The composition of  claim 132  which is rehydrated after lyophilization.  
     
     
         138 . The composition of  claim 132  which does not contain a disaccharide.  
     
     
         139 . A method of delivering polynucleotides to cells comprising: 
 contacting the composition of  claim 137  with cells, wherein the polynucleotide encodes a protein, whereby the protein is expressed.    
     
     
         140 . The composition of  claim 32  wherein said polycation is CK15-60P10, and the counterion is acetate, wherein CK15-60P10 is a polyamino acid polymer of one N-terminal cysteine and 15-60 lysine residues, wherein a molecule of polyethylene glycol having an average molecular weight of 10 kdal is attached to the cysteine residue.  
     
     
         141 . The composition of  claim 140  wherein the polycation molecule comprises 30 residues of lysine.  
     
     
         142 . The composition of  claim 140  wherein the polycation molecule comprises a targeting moiety.  
     
     
         143 . The composition of  claim 140  which is lyophilized.  
     
     
         144 . The composition of  claim 140  wherein said complex is compacted to a diameter which is less than double the theoretical diameter of a complex of said single nucleic acid and a sufficient number of positively charged residues to provide a charge ratio of about 1:1, in the form of a condensed sphere.  
     
     
         145 . The composition of  claim 140  which is rehydrated after lyophilization.  
     
     
         146 . The composition of  claim 140  which does not contain a disaccharide.  
     
     
         147 . A method of delivering polynucleotides to cells comprising: 
 contacting the compositions of  claim 145  with cells, wherein the polynucleotide encodes an antisense nucleic acid, whereby the antisense nucleic acid is expressed.    
     
     
         148 . The composition of  claim 35  wherein said polycation is CK15-60P10, and said counterion is acetate, wherein CK15-60P10 is a polyamino acid polymer of one N-terminal cysteine and 15-60 lysine residues, wherein a molecule of polyethylene glycol having an average molecular weight of 10 kdal is attached to the cysteine residue.  
     
     
         149 . The composition of  claim 148  wherein the polycation molecule comprises 30 residues of lysine.  
     
     
         150 . The composition of  claim 148  wherein the polycation molecule comprises a targeting moiety.  
     
     
         151 . The composition of  claim 148  which is lyophilized.  
     
     
         152 . The composition of  claim 148  which is lyophilized and rehydrated.  
     
     
         153 . The composition of  claim 148  which does not contain a disaccharide.  
     
     
         154 . A method of delivering polynucleotides to cells comprising: 
 contacting the composition of  claim 152  with cells, whereby the polynucleotide is delivered to and taken up by the cells.    
     
     
         155 . The method of  claim 41 , wherein said polycation is CK15-60P10, and said counterion is acetate, wherein CK15-60P10 is a polyamino acid polymer of one N-terminal cysteine and 15-60 lysine residues, wherein a molecule of polyethylene glycol having an average molecular weight of 10 kdal is attached to the cysteine residue.  
     
     
         156 . The method of  claim 155  further comprising lyophilizing the unaggregated nucleic acid complexes.  
     
     
         157 . The method of  claim 156  further comprising rehydrating the lyophilized nucleic acid complexes.  
     
     
         158 . The method of  claim 155  wherein the polycation molecule comprises 30 residues of lysine.  
     
     
         159 . The method of  claim 155  wherein the polycation molecule comprises a targeting moiety.  
     
     
         160 . A method of preparing a composition comprising unaggregated nucleic acid complexes, each complex consisting essentially of a single nucleic acid molecule and one or more polycation molecules, said method comprising: 
 mixing a nucleic acid molecule with a polycation molecule at a salt concentration sufficient for compaction of the complex to a diameter which is less than double the theoretical minimum diameter of a complex of said single nucleic acid molecule and a sufficient number of polycation molecules to provide a charge ratio of about 1:1, in the form of a condensed sphere, or 30 nm, whichever is larger, whereby unaggregated nucleic acid complexes are formed, wherein each complex consists essentially of a single nucleic acid molecule and one or more polycation molecules, and wherein said polycation molecules have a counterion selected from the group consisting of acetate, bicarbonate and chloride.    
     
     
         161 . The method of  claim 160  wherein the counterion is acetate.  
     
     
         162 . The method of  claim 160  wherein the polycation molecules are polylysine or a polylysine derivative.  
     
     
         163 . The method of  claim 162  wherein the polylysine derivative is polylysine peptide with a cysteine residue.  
     
     
         164 . Non-naturally occurring, soluble compacted complexes of a nucleic acid and a polycation molecule made by the method of  claim 160 .  
     
     
         165 . The method of  claim 160  wherein the salt is NaCl.  
     
     
         166 . The method of  claim 160  wherein the nucleic acid and the polycation are each, at the time of mixing, in a solution having a salt concentration of 0.05 to 1.5 M.  
     
     
         167 . The method of  claim 160  in which the mixing is monitored to detect, prevent or correct, the formation of aggregated or relaxed complexes.  
     
     
         168 . The method of  claim 160  in which the molar ratio of the phosphate groups of the nucleic acid to the positively charged groups of the polycation is in the range of 4:1 to 1:4.  
     
     
         169 . The method of  claim 160  in which the polycation is added to the nucleic acid, while vortexing at high speed.  
     
     
         170 . The method of  claim 160  in which the nucleic acid is added to the polycation, while vortexing at high speed.  
     
     
         171 . The method of  claim 160  wherein the mixing is monitored by a method selected from the group consisting of electron microscopy, light scattering, circular diochroism, and absorbance measurement.  
     
     
         172 . The method of  claim 160 , wherein said polycation is CK15-60P10 and the counterion is acetate, wherein CK15-60P10 is a polyamino acid polymer of one N-terminal cysteine and 15-60 lysine residues, wherein a molecule of polyethylene glycol having an average molecular weight of 10 kdal is attached to the cysteine residue.  
     
     
         173 . The method of  claim 172  further comprising lyophilizing the unaggregated nucleic acid complexes.  
     
     
         174 . The method of  claim 173  further comprising rehydrating the lyophilized nucleic acid complexes.  
     
     
         175 . The method of  claim 172  wherein the polycation molecule comprises 30 residues of lysine.  
     
     
         176 . The method of  claim 172  wherein the polycation molecule comprises a targeting moiety.  
     
     
         177 . The composition of  claim 2  wherein said polycation is CK30P5 or CK45P5 and the counterion is acetate, wherein CK30P5 or CK45P5 is a polyamino acid polymer of one N-terminal cysteine and 30 or 45 lysine residues, wherein a molecule of polyethylene glycol having an average molecular weight of 5 kdal is attached to the cysteine residue.  
     
     
         178 . The composition of  claim 20  wherein said polycation is CK30P5 or CK45P5 and the counterion is acetate, wherein CK30P5 or CK45P5 is a polyamino acid polymer of one N-terminal cysteine and 30 or 45 lysine residues, wherein a molecule of polyethylene glycol having an average molecular weight of 5 kdal is attached to the cysteine residue.  
     
     
         179 . The composition of  claim 29  wherein said polycation is CK30P5 or CK45P5 and the counterion is acetate, wherein CK30P5 or CK45P5 is a polyamino acid polymer of one N-terminal cysteine and 30 or 45 lysine residues, wherein a molecule of polyethylene glycol having an average molecular weight of 5 kdal is attached to the cysteine residue.  
     
     
         180 . The composition of  claim 32  wherein said polycation is CK30P5 or CK45P5 and the counterion is acetate, wherein CK30P5 or CK45P5 is a polyamino acid polymer of one N-terminal cysteine and 30 or 45 lysine residues, wherein a molecule of polyethylene glycol having an average molecular weight of 5 kdal is attached to the cysteine residue.  
     
     
         181 . The composition of  claim 35  wherein said polycation is CK30P5 or CK45P5 and the counterion is acetate, wherein CK30P5 or CK45P5 is a polyamino acid polymer of one N-terminal cysteine and 30 or 45 lysine residues, wherein a molecule of polyethylene glycol having an average molecular weight of 5 kdal is attached to the cysteine residue.  
     
     
         182 . The composition of  claim 41  wherein said polycation is CK30P5 or CK45P5 and the counterion is acetate, wherein CK30P5 or CK45P5 is a polyamino acid polymer of one N-terminal cysteine and 30 or 45 lysine residues, wherein a molecule of polyethylene glycol having an average molecular weight of 5 kdal is attached to the cysteine residue.  
     
     
         183 . The composition of  claim 160  wherein said polycation is CK30P5 or CK45P5 and the counterion is acetate, wherein CK30P5 or CK45P5 is a polyamino acid polymer of one N-terminal cysteine and 30 or 45 lysine residues, wherein a molecule of polyethylene glycol having an average molecular weight of 5 kdal is attached to the cysteine residue.  
     
     
         184 . The method of  claim 10  wherein the nucleic acid and the polycation are each, at the time of mixing, in a solution having a salt concentration of 0.0 M.  
     
     
         185 . The method of  claim 38  wherein the nucleic acid and the polycation are each, at the time of mixing, in a solution having a salt concentration of 0.0 M.  
     
     
         186 . The method of  claim 160  wherein the nucleic acid and the polycation are each, at the time of mixing, in a solution having a salt concentration of 0.0 M.