DESC:RELATED APPLICATIONS
This application is related to Indian provisional application IN202321011083 filed 18th Feb, 2023 and is incorporated herein in its entirely.
FIELD OF THE INVENTION
The present invention relates to polynucleotides comprising a codon optimized nucleotide sequence encoding human Factor IX, viral particles comprising the polynucleotides and treatments utilizing the polynucleotides.
BACKGROUND OF THE INVENTION
Haemophilia B, an X-linked life threatening bleeding disorder affects 1:30,000 males. Current treatment involves frequent intravenous injections (2-3 times per week) of human Factor IX (hFIX) protein. This treatment is highly effective at arresting bleeding but it is not curative and is extremely expensive (£150,000/patient/year), thus making it unaffordable by the majority of haemophilia B patients in the world. Gene therapy for haemophilia B offers the potential for a cure through persistent, endogenous production of human Factor IX following the transfer of a functioning copy of the human Factor IX gene to an affected patient.
The present application relates to a gene therapy approach for treating haemophilia B, involving administration of a vector comprising a polynucleotide encoding human Factor IX. Such a gene therapy approach would avoid the need for frequent intravenous injections of human factor IX. However, it is difficult to provide an effective gene therapy vector, i.e. one that allows a high level persistent expression of human Factor IX expression exhibiting high in vivo functionality/activity.
OBJECTS OF THE INVENTION
The main object of the present invention is to provide a polynucleotide comprising a human Factor IX nucleotide sequence, wherein the human Factor IX nucleotide sequence comprises a coding sequence that encodes a human Factor IX protein or fragment thereof and wherein a portion of the coding sequence is not wild type.
Another object of the present invention is to provide a codon optimized polynucleotide sequence encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 01, SEQ ID NO: 02; SEQ ID NO: 03; SEQ ID NO: 04 or SEQ ID NO: 05.
Another object of the present invention is to provide codon optimized polynucleotide encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identity to SEQ ID NO: 01.
Another object of the present invention is to provide codon optimized polynucleotide encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identity to SEQ ID NO: 02.
Another object of the present invention is to provide codon optimized polynucleotide encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identity to SEQ ID NO: 03.
Another object of the present invention is to provide codon optimized polynucleotide encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identity to SEQ ID NO: 04.
Another object of the present invention is to provide codon optimized polynucleotide encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identity to SEQ ID NO: 05.
Another object of the present invention is to provide a codon optimized polynucleotide sequence encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 01, SEQ ID NO: 02; SEQ ID NO: 03; SEQ ID NO: 04 or SEQ ID NO: 05, wherein said sequence is also atleast 50% codon optimized and 75% identical in compare with the wild type Factor IX nucleotide sequence having SEQ ID NO: 6.
Another object of the present invention is to provide a codon optimized polynucleotide sequence encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 01, SEQ ID NO: 02; SEQ ID NO: 03; SEQ ID NO: 04 or SEQ ID NO: 05, wherein said polynucleotide sequence further comprises any transcription regulatory element.
Another object of the present invention is to provide method of treatment of haemophilia comprising; administering an effective amount of the codon optimized polynucleotide sequence encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 01, SEQ ID NO: 02; SEQ ID NO: 03; SEQ ID NO: 04 or SEQ ID NO: 05.
SUMMARY OF THE INVENTION
The main aspect of the present invention is to provide a polynucleotide comprising a Factor IX nucleotide sequence, wherein the Factor IX nucleotide sequence comprises a coding sequence that encodes a human Factor IX protein or fragment thereof and wherein a portion of the coding sequence is not absolutely equivalent to wild type human Factor IX nucleotide.
Another aspect of the present invention is to provide a codon optimized polynucleotide sequence encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 01, SEQ ID NO: 02; SEQ ID NO: 03; SEQ ID NO: 04 or SEQ ID NO: 05.
Another aspect of the present invention is to provide codon optimized polynucleotide encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identity to SEQ ID NO: 01.
Another aspect of the present invention is to provide codon optimized polynucleotide encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identity to SEQ ID NO: 02.
Another aspect of the present invention is to provide codon optimized polynucleotide encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identity to SEQ ID NO: 03.
Another aspect of the present invention is to provide codon optimized polynucleotide encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identity to SEQ ID NO: 04.
Another aspect of the present invention is to provide codon optimized polynucleotide encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identity to SEQ ID NO: 05.
Another aspect of the present invention is to provide a codon optimized polynucleotide sequence encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 01, SEQ ID NO: 02; SEQ ID NO: 03; SEQ ID NO: 04 or SEQ ID NO: 05, wherein said sequence is codon optimized in compare with the wild type human Factor IX nucleotide sequence having SEQ ID NO: 06.
Another aspect of the present invention is to provide a codon optimized polynucleotide sequence encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 01, SEQ ID NO: 02; SEQ ID NO: 03; SEQ ID NO: 04 or SEQ ID NO: 05, wherein said sequence is also atleast 50% codon optimized and 75% identical in compare with the wild type human Factor IX nucleotide sequence having SEQ ID NO: 06.
Another aspect of the present invention is to provide a codon optimized polynucleotide sequence encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 01, SEQ ID NO: 02; SEQ ID NO: 03; SEQ ID NO: 04 or SEQ ID NO: 05, wherein said polynucleotide sequence further comprises any transcription regulatory element.
Another aspect of the present invention is to provide method of treatment of haemophilia comprising; administering an effective amount of the codon optimized polynucleotide sequence encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 01, SEQ ID NO: 02; SEQ ID NO: 03; SEQ ID NO: 04 or SEQ ID NO: 05.
BRIEF DESCRIPTION OF THE DRAWINGS
In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figure together with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure wherein:
Figure-1: The construction of an optimized liver-specific human factor IX (FIX) gene expression cassette and plasmid map of transfer plasmid.
Figure-2: Plasmid map of packaging plasmid.
Figure-3: Plasmid map of helper plasmid.
Figure-4: Characterization of pIntas-FIX-R338L-Kan-SQ5 Stbl3 Clones.
Figure-5: Restriction digestion analysis of pIntas-FIX-R338L-Kan-SQ5 plasmids isolated from P0 upon passaging of respective source cell bank.
Figure-6: Restriction digestion analysis of pIntas-FIX-R338L-Kan-SQ5 plasmids isolated from P2 upon passaging of respective source cell bank
Figure - 7: Restriction digestion analysis of pIntas-FIX-R338L-Kan-SQ5 plasmids isolated from P5 upon passaging of respective source cell bank
Figure - 8: Restriction digestion analysis of pIntas-FIX-R338L-Kan-SQ5 plasmids isolated from P6 upon passaging of respective source cell bank
Figure - 9: Restriction digestion analysis of pIntas-FIX-R338L-Kan-SQ5 plasmids isolated from P9 upon passaging of respective source cell bank
Figure - 10: Restriction digestion analysis of pIntas-FIX-R338L-Kan-SQ5 plasmids isolated from P11 upon passaging of respective source cell bank
Figure – 11: Schematic representation of Sequence alignment for pIntas-FIX-R338L-Kan-SQ5 Clone 5 P0 and P11 plasmids

DETAILED DESCRIPTION OF THE INVENTION
DEFINITION
The following definitions are provided to facilitate understanding of certain terms used throughout the specification.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs.
In general, the term “comprising” is intended to mean including but not limited to. For example, the phrase “a polynucleotide comprising a human Factor IX nucleotide sequence” should be interpreted to mean that the polynucleotide has a human Factor IX nucleotide sequence, but the polynucleotide may contain additional nucleotides.
In some embodiments of the invention, the word “comprising” is replaced with the phrase “consisting of”. The term “consisting of” is intended to be limiting. For example, the phrase “a polynucleotide consisting of a human Factor IX nucleotide sequence” should be understood to mean that the polynucleotide has a human Factor IX nucleotide sequence and no additional nucleotides.
The terms “protein” and “polypeptide” are used interchangeably herein, and are intended to refer to a polymeric chain of amino acids of any length.
The term “polynucleotide” refers to a polymeric form of nucleotides of any length, deoxyribonucleotides, ribonucleotides, or analogs thereof. For example, the polynucleotide may comprise DNA (deoxyribonucleotides) or RNA (ribonucleotides). The polynucleotide may consist of DNA. The polynucleotide may be mRNA. Since the polynucleotide may comprise RNA or DNA, all references to T (thymine) nucleotides may be replaced with U (uracil).
A “coding sequence” is a sequence that encodes a polynucleotide, and excludes non coding regions such as introns. A coding sequence may be interrupted by non-coding nucleotides (e.g. an intron), but only nucleotides that encode the polypeptide should be considered to be part of the coding sequence. For example, a coding sequence that encodes a human Factor IX protein will comprise any codons that encode an amino acid forming part of the human Factor IX protein that is expressed from that coding sequence, irrespective of whether those codons are contiguous in sequence or separated by one or more non-coding nucleotides. In other words, a polynucleotide which contains stretches of coding nucleotides interrupted by a stretch of non-coding nucleotides will be considered to comprise a “coding sequence” consisting of the non-contiguous coding stretches immediately juxtaposed (i.e. minus the non-coding stretch). However, herein, the stop codon will be considered to be part of the full length coding sequence. The term “sequence that encodes” refers to a nucleotide sequence comprising codons that encode the encoded polypeptide.
The main embodiment of the present invention is to provide a polynucleotide comprising a human Factor IX nucleotide sequence, wherein the Factor IX nucleotide sequence comprises a coding sequence that encodes a human Factor IX protein or fragment thereof and wherein a portion of the human Factor IX nucleotide sequence is not wild type.
The polynucleotide may further comprise one or more of the following features. The polynucleotide may comprise a portion that is not codon optimized. The polynucleotide may comprise an intron or a fragment of an intron. The polynucleotide may comprise a mutation in a codon corresponding to codon 384 of wild type human Factor IX.
Another embodiment of the present invention is to provide a codon optimized polynucleotide sequence encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 01, SEQ ID NO: 02; SEQ ID NO: 03; SEQ ID NO: 04 or SEQ ID NO: 05.
Another embodiment of the present invention is to provide codon optimized polynucleotide encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identity to SEQ ID NO: 01.
Another embodiment of the present invention is to provide codon optimized polynucleotide encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identity to SEQ ID NO: 02.
Another embodiment of the present invention is to provide codon optimized polynucleotide encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identity to SEQ ID NO: 03.
Another embodiment of the present invention is to provide codon optimized polynucleotide encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identity to SEQ ID NO: 04.
Another embodiment of the present invention is to provide codon optimized polynucleotide encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identity to SEQ ID NO: 05.
Another embodiment of the present invention is to provide a codon optimized polynucleotide sequence encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 01, SEQ ID NO: 02; SEQ ID NO: 03; SEQ ID NO: 04 or SEQ ID NO: 05, wherein said sequence is codon optimized in compare with the wild type human Factor IX nucleotide sequence having SEQ ID NO: 06.
Another embodiment of the present invention is to provide a codon optimized polynucleotide sequence encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 01, SEQ ID NO: 02; SEQ ID NO: 03; SEQ ID NO: 04 or SEQ ID NO: 05, wherein said sequence is also at least 50% codon optimized and 75% identical in compare with the wild type human Factor IX nucleotide sequence having SEQ ID NO: 06.
Another embodiment of the present invention is to provide a codon optimized polynucleotide sequence encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 01, SEQ ID NO: 02; SEQ ID NO: 03; SEQ ID NO: 04 or SEQ ID NO: 05, wherein said polynucleotide sequence further comprises any transcription regulatory element.
In another embodiment of the present invention the transcription regulatory element may comprise a promoter and/or an enhancer, such as the promoter element and/or enhancer element from HLP2, HLP1, LP1, HCR-hAAT, ApoE-hAAT, and LSP. Each of these transcription regulatory elements comprises a promoter, an enhancer, and optionally other nucleotides.
Another embodiment of the present invention is to provide method of treatment of haemophilia comprising; administering an effective amount of the codon optimized polynucleotide sequence encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 01, SEQ ID NO: 02; SEQ ID NO: 03; SEQ ID NO: 04 or SEQ ID NO: 05.
In an embodiment, the human Factor IX protein or fragment thereof comprises a mutation at a position corresponding to position 384 of wild type human Factor IX. For example, position 384 (numbering from the start of the signal peptide, i.e. a position corresponding to amino acid 384 of SEQ ID NO: 08) of wild type human Factor IX is an arginine residue (R384), but this can be replaced by a different residue. In an embodiment, R384 is replaced with a small, hydrophobic amino acid. For example, the small, hydrophobic amino acid could be alanine, isoleucine, leucine, valine or glycine. Preferably, the human Factor IX protein or fragment thereof comprises a leucine at a position corresponding to position 384 in wild type human Factor IX, as shown in SEQ ID NO: 08. A mutation at a position corresponding to position 384 of the wild type sequence may cause a gain-of-function (GoF) mutation, resulting in human Factor IX that is hyperfunctional. The advantage of expressing a human Factor IX protein containing a mutation at position 384 is that a relatively small increase in protein amount produces a larger increase in overall protein activity.
It is within the abilities of the person skilled in the art to determine whether a given polypeptide has a mutation at a position corresponding to position 384. The person skilled in the art merely needs to align the sequence of the polypeptide sequence with that of a wild type (precursor, immature) human Factor IX polypeptide, and determine whether the residue of the former that aligns with the 384th residue of the latter is an arginine. If not, the polypeptide has a mutation at a position corresponding to position 384 in wild type human Factor IX.
The wildtype sequence of polynucleotide encoding human Factor IX is disclosed in WO 2020/039183.
Another variant of human Factor IX, 194T/A (ACT/GCT) is a common natural variant present in human factor IX which is disclosed in Zacchi et. al., Nat. Commun Biol., 2021, originated due to presence of single nucleotide polymorphism (580A/G).
In another embodiment of the present invention any of the wild type human Factor IX referenced above can be used.
The present application discloses that certain codons are favoured for expression in the human liver and that reducing the CpG content of a coding sequence, whilst maintaining a high proportion of those favoured codons, improves expression of the coding sequence. The favoured codons are TTC, CTG, ATC, GTG, GTC, AGC, CCC, ACC, GCC, TAC, CAC, CAG, AAC, AAA, AAG, GAC, TGC, AGG, GGC, and GAG.
In one embodiment, the portion of the coding sequence that is codon optimized is codon optimized for human expression including liver and includes a higher proportion of codons, such as codons TTC, CTG, ATC, GTG, GTC, AGC, CCC, ACC, GCC, TAC, CAC, CAG, AAC, AAA, AAG, GAC, TGC, AGG, GGC, and GAG.
In an embodiment, the portion of the coding sequence that is codon optimized comprises codons that encode phenylalanine, leucine, isoleucine, valine, serine, proline, threonine, alanine, tyrosine, histidine, glutamine, asparagine, lysine, aspartate, glutamate, cysteine, tryptophan, arginine, and glycine.
In another embodiment the polynucleotide sequence encoding FIX protein which is optimized has at least 80% of the codons are selected from the group consisting of:
1. GCT and/or GCC encoding alanine,
2. TGC encoding cysteine,
3. GAC and or GAT encoding aspartic acid
4. GAG and GAA encoding glutamic acid
5. TTC and/or TTT encoding phenylalanine
6. GGC, GGA and/or GGG encoding glycine
7. CAC encoding histidine
8. ATC encoding isoleucine
9. AAA and/or AAG encoding lysine
10. CTG and/or CTC encoding leucine
11. AAT and/or AAC encoding asparagine
12. CCT and/or CCC encoding proline
13. CAG encoding glutamine
14. AGA and/or AGG encoding arginine
15. TCT, TCC and/or AGC encoding serine
16. ACC and/or ACA encoding threonine
17. GTG and/or GTC encoding valine
18. TAC encoding tyrosine
In another embodiment the polynucleotide sequence encoding FIX protein which is optimized has at least following codons:
1. 87% codons that encode alanine are GCT and GCC,
2. 43% codons that encode alanine are GCC,
3. 54% codons that encode cysteine are TGC,
4. 58% codons that encode aspartic acid are GAC
5. 56% codons that encode glutamic acid are GAG
6. 33% codons that encode phenylalanine are TTC or TTT
7. 86% codons that encode glycine are GGC, GGA and GGG
8. 39% codons that encode glycine is GGC
9. 22% codons that encode glycine is GGA
10. 50% codons that encode histidine are CAC
11. 24% codons that encode isoleucine are ATT or ATC
12. 29% codons that encode lysine are AAG or AAA
13. 07% codons that encode leucine are CTC
14. 59% codons that encode leucine are CTG
15. 56% codons that encode asparagine are AAC
16. 73% codons that encode proline are CCT and CCC
17. 27% codons that encode proline is CCC
18. 79% codons that encode glutamine are CAG
19. 74% codons that encode arginine are AGA and AGG
20. 11% codons that encode arginine is AGG
21. 67% codons that encode serine are TCT, TCC and AGC
22. 19% codons that encode serine is AGC
23. 77% codons that encode threonine are ACC and ACA
24. 37% codons that encode threonine are ACC
25. 86% codons that encode valine are GTG and GTC
26. 16% codons that encode valine are GTC
27. 63% codons that encode tyrosine are TAC
In another embodiment portion that is codon optimized has same number of CpG dipeptides as wild type sequence.
In another embodiment portion that is codon optimized has greater number of CpG dipeptides as wild type sequence.
In another embodiment portion that is codon optimized has less number of CpG dipeptides as wild type sequence.
In another embodiment human Factor IX protein of the present invention has activity greater than the activity of wild type human Factor IX protein.
Another embodiment of the present invention is provide expression cassette comprising 5’ITR, LP1 enhancer/promoter, and codon optimized FIX-R338L and SV40 polyadenylation sequence.
Another embodiment of the present invention is provide expression cassette comprising 5’ITR, LP1 enhancer/promoter, and codon optimized FIX-R338L sequence.
Another object of the present invention is to provide expression cassette for human Factor IX comprising polynucleotide sequence having SEQ ID NO: 8 wherein, Factor IX polynucleotide sequence within said expression cassette is selected from SEQ ID NO: 1, SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04 or SEQ ID NO: 05.
Another object of the present invention is to provide packaging plasmid comprising SEQ ID NO: 9.
Another object of the present invention is to provide helper plasmid comprising a nucleotide sequence encoding at least one essential function required for the replication and stability of a recipient plasmid, wherein the helper plasmid is capable of providing the essential function to the recipient plasmid in a host cell.
Another object of the present invention is to provide helper plasmid comprising a nucleotide sequence encoding Adenovirus VA, adenovirus E4 and adenovirus E2A.
The embodiments of the present invention are further described using specific examples herein after. The examples are provided for better understanding of certain embodiments of the invention and not, in any manner, to limit the scope thereof. Possible modifications and equivalents apparent to those skilled in the art using the teachings of the present description and the general art in the field of the invention shall also from the part of this specification and are intended to be included within the scope of it.

EXPERIMENT DETAILS
EXAMPLE: 1 IN SILICO CONSTRUCT DESIGN
A total of 5 constructs were designed in silico with varying levels of codon optimization. One of the constructs - FIX-R338L-SQ5 were chosen for further consideration based on the optimum extent of codon optimization and minimal number of CpG region. Details of the constructs are provided in table 1.
Table 1: Details of the constructs
Seq ID 1 2 3 4 5
CodOp (397) 218 228 218 217 212
% CodOp 55.1 57.6 55.1 54.8 53.5
Nt identity (939/1185) (923/1185) (936/1185) (937/1185) (937/1185)
CpG (wt-15) 27 28 28 18 16

The full construct of FIX-R338L-SQ5 was synthesized from GenScript. The plasmid obtained was transformed in E.coli Stbl3 strain and plated on Kanamycin containing LBV agar plate. The plasmid has the kanamycin resistance gene and the colonies harboring the plasmid would grow on the plate in colonies, upon overnight incubation. Six colonies were picked up from the plate and grown in Kanamycin containing LBV medium for plasmid isolation. The plasmid was then digested using construct-specific restriction endonucleases and ran on agarose gel, to ensure its identity and integrity. Refer to Table-1 for enzymes used and bands obtained.
Table 2: Restriction enzymes used for clone characterization
Sr. No. Plasmid Name Restriction Enzyme for linearization Restrictions Enzyme(s) for multiple digestion
1 pIntas-FIX-R338L-Kan-SQ5 MfeI (5033 bp) MfeI + EcoRI (2764 bp +1483 bp + 786 bp)

EXAMPLE 2: STABILIY ANALYSIS OF FIX ENCODING CLONES
Best 3 clones (Clone no- 3, 4 and 5) out of total 6 colonies picked were selected for stability analysis, based on quality of band pattern obtained in restriction digestion analysis (Figure-4 & Table-3). Clone stability analysis was performed by subsequently passaging the thawed glycerol stock of the plasmid up to 11 passages. Plasmid was extracted at passage P0, P2, P5, P6, P9 and P11. All these plasmid were subjected for restriction digestion analysis (Figure- 5 to 10).
Based on our plasmid stability data, clone 4 and 5 were found to be stable over all the 11 passages, with only the expected bands appearing on the gel with no additional unexplained bands (Figure-5 to 10). Clone-5 was chosen further subjected to sequencing verification at passage P0 and P11 (Figure-11).
Table 3: Plasmid concentration of Stbl3 clones of pIntas-FIX-R338L-Kan-SQ5
Sr. No. Clone ID Concentration of DNA (ng/µL) A260/A280 A260/A230
1 pIntas-FIX-R338L-Kan-SQ5 Stbl3 Clone 1 220.6 1.88 2.14
2 pIntas-FIX-R338L-Kan-SQ5 Stbl3 Clone 2 251.2 1.88 2.30
3 pIntas-FIX-R338L-Kan-SQ5 Stbl3 Clone 3 293.7 1.89 2.30
4 pIntas-FIX-R338L-Kan-SQ5 Stbl3 Clone 4 266.0 1.89 2.28
5 pIntas-FIX-R338L-Kan-SQ5 Stbl3 Clone 5 292.2 1.89 2.30
6 pIntas-FIX-R338L-Kan-SQ5 Stbl3 Clone 6 246.7 1.88 2.27
Table 4: OD600 and plasmid DNA content at different passages for pIntas-FIX-R338L-Kan-SQ5
Passage No. Clone 3 Clone 4 Clone 5
O.D.600 DNA Conc. (ng/µL) DNA Conc./1 Unit O.D.600 O.D.600 DNA Conc. (ng/µL) DNA/1 Unit O.D.600 O.D.600 DNA Conc. (ng/µL) DNA/1 Unit O.D.600
P0 4.1 89.6 17.92 2.9 101.5 20.30 2.9 99.1 19.82
P1 4.2 69.1 13.82 5.2 92.5 18.50 5.0 80.6 16.12
P2 4.9 116.5 23.30 4.2 107.8 21.56 4.5 86.9 17.38
P3 3.6 81.4 16.28 5.7 90.8 18.16 4.0 81.3 16.26
P4 9.0 117 23.40 10.7 183.3 36.66 13.5 168.3 33.66
P5 10.7 124.4 24.88 7.1 174.5 34.90 14.2 110.4 22.08
P6 7.6 131.2 26.24 8.0 98.4 19.68 12.2 99 19.80
P7 7.6 220.2 44.04 8.0 205.1 41.02 12.2 137.7 27.54
P8 9.6 180.9 36.18 10.9 170.8 34.16 10.4 173.5 34.70
P9 10.1 94.4 18.88 14.3 153.3 30.66 7.1 154.9 30.98
P10 13.3 157.3 31.46 9.3 188.4 37.68 9.0 210.6 42.12
P11 5.9 105.6 21.12 5.4 123.4 24.68 5.9 175.8 35.16

EXAMPLE 3: PRODUCTION OF AAV8-FIX R338L IN SHAKE FLASK
Evaluation of pFIX-R338L plasmid was done in the viral vector production batch using HEK293SF 3F6 suspension cell line. Intensified Culture of HEK293SF 3F6 was used for seeding the production shake flasks (in duplicate) targeting 2.0 -2.5 million cells/mL cell density. Post reaching the cell count 3.5 – 4.5 million cells/mL in the production shake flask, transfection was performed using pFIX-R338L:pR2C8: pHelper plasmids. 72-hour post transfection harvest, Lysis and clarification was done. Estimated CP/mL and VG/mL titers are mentioned in table 5.
Table 5: Estimated CP/mL and VG/mL AAV8 titers for pIntas-FIX-R338L-Kan-SQ5 produced in HEK293SF 3F6 cells
Sr. No. Sample No./ Sample name CP/mL VG/mL
1. (Production Shake Flask 1) 3.70E+11 4.29E+10
2. (Production Shake Flask 2) 3.52E+11 3.75E+10

EXAMPLE 4: EXPESSION OF FIX R338L IN AAV8-FIX R338L TRANSDUCED HEPG2 CELLS
The potency of the FIX Padua GTP was determined using transduction assay. HepG2 cells (a cell line originated from liver) at different cell densities were transduced at MOI of 5x105 with FIX Padua gene therapy product. Cells were given a media change 18-24 hours post transduction. Transduction supernatants were collected on Day 5 post media change and FIX secretion quantitated using FIX Ag ELISA kit.
Table 6: Expression of recombinant FIX-R338L from AAV8-FIX-R338L transduced HEPG2 cells in different cell densities
Cell Seeding Density MOI Factor IX antigen level (IU/mL)
5.00E+05 5.00E+05 1.194
2.50E+05 0.758
1.00E+05 0.170 ,CLAIMS:We Claim,
1. A polynucleotide comprising a human Factor IX nucleotide sequence, wherein the Factor IX nucleotide sequence comprises: a coding sequence that encodes a human Factor IX protein or fragment thereof and wherein a portion of the human Factor IX nucleotide sequence is not wild type.
2. The polynucleotide sequence according to claim 1, wherein said human Factor IX sequence is codon optimized in compare with the wild type human Factor IX nucleotide sequence having SEQ ID NO: 06.
3. The polynucleotide sequence according to claim 1, wherein said human Factor IX sequence is at least 50% codon optimized and 75% identical in compare with wild type human Factor IX nucleotide sequence having SEQ ID NO: 06.
4. The polynucleotide sequence according to claim 1, wherein said human Factor IX sequence comprises: promoter, an enhancer, and optionally other nucleotide.
5. A codon optimized polynucleotide sequence encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 90% identical to SEQ ID NO: 01, SEQ ID NO: 02, SEQ ID NO:03; SEQ ID NO:04 or SEQ ID NO:05.
6. A method of treatment of haemophilia comprising: administering an effective amount of the codon optimized polynucleotide sequence encoding human Factor IX protein, wherein said polynucleotide sequence is at least 90% identical to SEQ IDNO: 01, SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04 or SEQ ID NO: 05.
7. The polynucleotide sequence according to any of the previous claims, wherein said codon optimized sequence has at least 80% of the codons selected from the group consisting of:
a. GCT and/or GCC encoding alanine,
b. TGC encoding cysteine,
c. GAC and or GAT encoding aspartic acid
d. GAG and GAA encoding glutamic acid
e. TTC and/or TTT encoding phenylalanine
f. GGC, GGA and/or GGG encoding glycine
g. CAC encoding histidine
h. ATC encoding isoleucine
i. AAA and/or AAG encoding lysine
j. CTG and/or CTC encoding leucine
k. AAT and/or AAC encoding asparagine
l. CCT and/or CCC encoding proline
m. CAG encoding glutamine
n. AGA and/or AGG encoding arginine
o. TCT, TCC and/or AGC encoding serine
p. ACC and/or ACA encoding threonine
q. GTG and/or GTC encoding valine
r. TAC encoding tyrosine
8. A codon optimized polynucleotide sequence encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 90% identical to SEQ ID NO: 05.
9. A codon optimized polynucleotide encoding human Factor IX protein, wherein the codon optimized polynucleotide sequence is at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identity to SEQ ID NO: 05.