METHODS FOR FORMING POLYPLEXES

1 . A method for making one or more polyplexes, the method comprising:

(a) providing:

i. a polymer in a first liquid stream;

ii. a nucleic acid component in a second liquid stream;

(b) contacting the polymer in the first liquid stream with the nucleic acid component in the second liquid stream to form a polyplex having a size and charge that is suitable for therapeutic administration; and

(c) isolating the polyplex to provide a stabilized polyplex.

2 . The method of claim 1 , wherein the isolating comprises conveying the polyplex in a liquid channel for a residence time sufficient to stabilize the polyplex.

3 . The method of any one of the preceding claims, further comprising assessing and harvesting one or more polyplexes.

4 . The method of any one of the preceding claims, wherein the first liquid stream and first liquid streams are solutions.

5 . The method of any one of the preceding claims, further comprising flowing the first liquid stream and the second liquid stream through a flow-regulating device at a rate that provides a polyplex having a size and charge that is suitable for transdermal administration.

6 . The method of claim 5 , wherein flow-regulating device is selected from the group consisting of a positive displacement pump, a syringe driven pump, a pressure driven pump, and a gravity feed pump.

7 . The method of any one of the preceding claims, wherein the contacting step uses a nozzle, a micro fluidics mixing device, a touch tube, a liquid bridge, a vertical mixer, a rotating double tube, or an atomizer.

8 . The method any one of the preceding claims, wherein the contacting step occurs in an air gap or in carrier fluid within the liquid channel.

9 . The method of any one of the preceding claims, wherein the stabilized polyplex is slightly positively charged.

10 . The method of any one of claims 2 - 9 , wherein the liquid channel is slightly negatively charged.

11 . The method of claim 10 , wherein the liquid channel comprises an aqueous phase surrounded by a carrier fluid.

12 . The method of claim 11 , wherein the density of the carrier fluid is a value in the range of 1,300 to 2,000 kg/m 3 and the density of the aqueous phase is a value in the range of 900 to 1200 kg/m 3 .

13 . The method of claim 12 , wherein the aqueous phase comprises sodium acetate buffer.

14 . The method of any claim 11 , wherein the carrier fluid is an oil.

15 . The method of claim 14 , wherein the oil is selected from the group consisting of: (a) Fluorinert FC-40 (fluorocarbonated oil), (b) silicon oil, (c) mineral oil, (d) perfluorinated amine oil, (e) phenylmethylpolysiloxane and (f) phenylmethylpolysiloxane-based oil and a additive.

16 . The method of claim 15 , wherein the additive has a hydrophilic-lipophilic balance number in the range of 2 to 8.

17 . The method of claim 15 , wherein the additive is a polysorbate additive.

18 . The method of claim 17 , wherein the polysorbate additive is SPAN 80, SPAN 65 or Tween 20.

19 . The method of claim 15 , wherein the concentration of the additive in the aqueous phase is from about 0.001% and about 10% (wt/wt %).

20 . The method of any one of any one of the preceding claims, wherein the isolating comprises segregating the polyplexes in the liquid channel.

21 . The method of claim 20 , wherein the space between the segregated polyplexes is controlled by the velocity of liquid in the channel.

22 . The method of any one of claims 2 - 21 , wherein the residence time is from about 1 second to about 20 minutes.

23 . The method of claim 22 , wherein the residence time is about 10 minutes.

24 . The method of any one of claims 3 - 23 , wherein assessing comprises:

measuring the refractive index of polyplexes in the liquid channel; and

optionally removing polyplexes with a refractive index which does not conform to specification.

25 . The method of any one of the preceding claims, further comprising filtering, washing, freezing and/or lyophilizing the stabilized polyplexes.

26 . The method of any one of the preceding claims, wherein the polymer is positively charged and the nucleic acid component is negatively charged.

27 . The method of any one of the preceding claims, wherein the polymer and nucleic acid component are mixed in a ratio of from about 0.1:1 to about 200:1 (w/w).

28 . The method of claim 27 , wherein the polymer and the nucleic acid component are mixed in a ratio of from about 20:1 to about 80:1 (w/w).

29 . The method of claim 27 , wherein the polymer and the nucleic acid component are mixed in a ratio of about 30:1 (w/w).

30 . The method of any one of the preceding claims, wherein the polymer is an HPAE.

31 . The method of any one of the preceding claims, wherein the polymer and nucleic acid component are present at a ratio of from about 0.1:1 to about 200:1 (w/w) in the polyplex.

32 . The method of claim 31 , wherein polymer and nucleic acid component are present at a ratio of from about 20:1 to about 80:1 (w/w) in the polyplex.

33 . The method of claim 31 , wherein polymer and nucleic acid component are present at a ratio of about 30:1 (w/w) in the polyplex.

34 . The method of any one of the preceding claims, wherein the particle size of the stabilized polyplex is less than about 2 μm.

35 . The method of claim 34 , wherein the particle size of the stabilized polyplex is about 60 nm to about 250 nm.

36 . The method of claim 34 , wherein the particle size of the stabilized polyplex is about 175 nm to about 250 nm.

37 . The method of any one of the preceding claims, wherein the zeta potential of the stabilized polyplex is from about 0 mV to about 100 mV.

38 . The method of claim 37 , wherein the zeta potential of the stabilized polyplex is from about 30 mV to about 34 mV.

39 . The method of any one of the preceding claims, wherein the stabilized polyplex is spherical.

40 . The method of any one of the preceding claims, wherein the polymer has a Mw of about 10 kDa.

41 . The method of any one of the preceding claims, wherein the nucleic acid component is a plasmid, nanoplasmid, nucleic acid, minicircle, or gene editing system.

42 . The method of claim 41 , wherein the nucleic acid component comprises a gene associated with a genetic disease or disorder.

43 . The method of claim 42 , wherein the genetic disease or disorder is caused by a mutation in one or more genes that results in low, absent, or dysfunctional protein expression.

44 . The method of claim 43 , wherein the gene is selected from the group consisting of COL7A1, LAMB3, ADA, SERPINA1, CFTR, HTT, NF1, PHA, HBS, FERMT1, KRT14, DSP, SPINK5, and FLG.

45 . The method of claim 44 , wherein the gene is COL7A1 and the genetic disease or disorder is a form of epidermolysis bullosa.

46 . The method of claim 45 , wherein the sequence of the gene is optimized for maximum protein expression upon delivery of the polyplex to a cell.

47 . The method of any one of the preceding claims, wherein the polymer has an alpha parameter defined from the Mark-Houwink equation of less than about 0.5.

48 . The method of any one of the preceding claims, wherein the polymer has an alpha parameter defined from the Mark-Houwink equation from about 0.2 to about 0.5.

49 . The method of any one of the preceding claims, wherein the polymer has a PDI from about 1.01 to about 8.0.

50 . The method of any one of the preceding claims, wherein the polymer has a PDI of about 2.5.

51 . The method of any one of the preceding claims, wherein the polymer has a Mw of at least 3 kDa.

52 . The method of any one of the preceding claims, wherein the polymer has a Mw of between about 5 kDa and 50 kDa.

53 . The method of any one of the preceding claims, wherein the polymer has a Mw of about 10 kDa.