WO2024035649A1

WO2024035649A1 - Compositions for progranulin expression and methods of use thereof

Info

Publication number: WO2024035649A1
Application number: PCT/US2023/029654
Authority: WO
Inventors: Richard Sullivan; Samantha Robin EDWARDS; Jacob Michael TOME; Jennifer Eunmi MOON
Original assignee: Shape Therapeutics Inc.
Priority date: 2022-08-08
Filing date: 2023-08-07
Publication date: 2024-02-15

Abstract

Described herein are recombinant polynucleotides comprising a promoter operably linked to a sequence encoding a payload. In some embodiments, the payload is progranulin and the sequence encoding the payload is a GRN sequence encoding progranulin. Described herein are recombinant polynucleotides comprising an intron for modulating expression of human progranulin. Also described herein are methods of treating a disease or disorder using the recombinant polynucleotide, such as treating frontotemporal dementia.

Description

COMPOSITIONS FOR PROGRANULIN EXPRESSION AND METHODS OF USE

THEREOF

CROSS-REFERENCE

[0001] The present application claims the benefit of U.S. Provisional Application No. 63/396,200, entitled “COMPOSITIONS FOR PROGRANULIN EXPRESSION AND METHODS OF USE THEREOF,” filed on August 8, 2022, and U.S. Provisional Application No. 63/421,358, entitled “COMPOSITIONS FOR PROGRANULIN EXPRESSION AND METHODS OF USE THEREOF,” filed on November 1, 2022, which applications are each herein incorporated by reference in their entireties for all purposes.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in extensible Markup Language (XML) format and is hereby incorporated by reference in its entirety. Said XML copy, created on August 7, 2023, is named “421688- 715021_SL.xml” and is 246 kilobytes in size.

BACKGROUND

[0003] A wide variety of diseases and disorders are caused by mutations, deletions, or altered expression of genes. For example, these mutations, deletions, or altered expression of genes causes under-expression, or low or no functionality the protein expressed from the gene leading to these diseases or disorders. Administration of gene therapy to individuals with missing, mutated, or improperly expressed genes may provide a useful means for treating such diseases and disorders.

SUMMARY

[0004] In various aspects, the present disclosure provides a recombinant polynucleotide comprising: an RSV promoter; a coding sequence operably linked to the RSV promoter, wherein the coding sequence encodes a progranulin; a post-transcriptional regulatory element; a polyadenylation signal; and a transcriptional pause site.

[0005] In some aspects, the post-transcriptional regulatory element comprises a WPRE. In some aspects, the WPRE is a shortened WPRE. In some aspects, the shortened WPRE is a WPRE3 comprising at least at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 8. In some aspects, the WPRE3 comprises at least 90% sequence identity to SEQ ID NO: 8. In some aspects, the WPRE3 comprises a sequence of SEQ ID NO: 8. In some aspects, the WPRE comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 7. In some aspects, the WPRE comprises at least 90% sequence identity to SEQ ID NO: 7. In some aspects, the WPRE comprises a sequence of SEQ ID NO: 7.

[0006] In some aspects, the post-transcriptional regulatory element is downstream of the coding sequence. In some aspects, the polyadenylation signal comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 42, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 43 - SEQ ID NO: 48, or SEQ ID NO: 55. In some aspects, the polyadenylation signal comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 42. In some aspects, the polyadenylation signal comprises at least 90% sequence identity to SEQ ID NO: 42. In some aspects, the polyadenylation signal comprises a sequence of SEQ ID NO: 42. In some aspects, the polyadenylation signal is downstream of the coding sequence.

[0007] In some aspects, the transcriptional pause site comprises a second polyadenylation signal. In some aspects, the transcriptional pause site comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 64. In some aspects, the transcriptional pause site comprises at least 90% sequence identity to SEQ ID NO: 64. In some aspects, the transcriptional pause site comprises a sequence of SEQ ID NO: 64. In some aspects, the transcriptional pause site is downstream of the coding sequence. In some aspects, the transcriptional pause site is downstream of the polyadenylation signal.

[0008] In some aspects, the coding sequence is downstream of the RSV promoter, the post- transcriptional regulatory element is downstream of the coding sequence, the polyadenylation signal is downstream of the post-transcriptional regulatory element, and the transcriptional pause site is downstream of the post-transcriptional regulatory element. In some aspects, the post- transcriptional regulatory element is downstream of the coding sequence, the polyadenylation signal is downstream of the post-transcriptional regulatory element, and the transcriptional pause site is downstream of the polyadenylation signal.

[0009] In some aspects, the RSV promoter comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 3. In some aspects, the RSV promoter comprises a sequence of SEQ ID NO: 3.

[0010] In some aspects, the progranulin is a human progranulin. In some aspects, the progranulin encoded by the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 63. In some aspects, the progranulin coding sequence codes for an amino acid sequence comprising the sequence of SEQ ID NO: 63. In some aspects, the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 10. In some aspects, the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 6. In some aspects, the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 11. In some aspects, the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 26. In some aspects, the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 57 and at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 58.

[0011] In some aspects, the recombinant polynucleotide further comprises a stuffer sequence. In some aspects, the stuffer sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 61. In some aspects, the stuffer sequence is upstream of the coding sequence. In some aspects, the stuffer sequence comprises a sequence of SEQ ID NO: 61. In some aspects, the stuffer sequence is upstream of the RSV promoter. In some aspects, the stuffer sequence is upstream of the RSV promoter, the coding sequence is downstream of the RSV promoter, the post-transcriptional regulatory element is downstream of the coding sequence, the polyadenylation signal is downstream of the post-transcriptional regulatory element, and the transcriptional pause site is downstream of the polyadenylation signal.

[0012] In some aspects, the recombinant polynucleotide comprises a 5’ untranslated region. In some aspects, the 5’ untranslated region comprises a Kozak sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 66. In some aspects, the 5’ untranslated region comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 56. In some aspects, The recombinant polynucleotide of any one of claims 41-43, wherein the 5’ untranslated region comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 62. In some aspects, the 5’ untranslated region comprises a sequence of SEQ ID NO: 62In some aspects, the 5’ untranslated region is upstream of the coding sequence. In some aspects, the 5’ untranslated region is downstream of the RSV promoter. In some aspects, the stuffer sequence is upstream of the RSV promoter, the 5 ’ untranslated region is downstream of the RSV promoter, the coding sequence is downstream of the 5 ’ untranslated region, the post-transcriptional regulatory element is downstream of the coding sequence, the polyadenylation signal is downstream of the post- transcriptional regulatory element, and the transcriptional pause site is downstream of the polyadenylation signal.

[0013] In some aspects, the recombinant polynucleotide comprises a 5’ inverted terminal repeat upstream of the RSV promoter and a 3’ inverted terminal repeat downstream of the transcriptional pause site. In some aspects, the recombinant polynucleotide further comprises a 5 ’ inverted terminal repeat upstream of the stuffer sequence and a 3 ’ inverted terminal repeat downstream of the transcriptional pause site. In some aspects, the recombinant polynucleotide further comprises a 5 ’ inverted terminal repeat and a 3 ’ inverted terminal repeat, wherein the 5 ’ inverted terminal repeat is upstream of the stuffer sequence, the stuffer sequence is upstream of the RSV promoter, the 5 ’ untranslated region is downstream of the RSV promoter, the coding sequence is downstream of the 5 ’ untranslated region, the post-transcriptional regulatory element is downstream of the coding sequence, the polyadenylation signal is downstream of the post- transcriptional regulatory element, the transcriptional pause site is downstream of the polyadenylation signal, and the 3 ’ inverted terminal repeat downstream of the transcriptional pause site. In some aspects, the 5’ inverted terminal repeat is an AAV 5’ inverted terminal repeat. In some aspects, the AAV 5 ’ inverted terminal repeat is a single stranded AAV 5 ’ inverted terminal repeat. In some aspects, the 5’ inverted terminal repeat is an AAV5 5’ inverted terminal repeat, an AAV1 5’ inverted terminal repeat, an AAV2 5’ inverted terminal repeat, an AAV9 5’ inverted terminal repeat, or a PhP.eB 5’ inverted terminal repeat. In some aspects, the 5’ inverted terminal repeat is an AAV2 5’ inverted terminal repeat. In some aspects, the 5’ inverted terminal repeat comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 59. In some aspects, the 5’ inverted terminal repeat comprises a sequence of SEQ ID NO: 59. In some aspects, the 3’ inverted terminal repeat is an AAV 3’ inverted terminal repeat. In some aspects, the AAV 3’ inverted terminal repeat is a single stranded AAV 3 ’ inverted terminal repeat. In some aspects, the 3’ inverted terminal repeat is an AAV5 3’ inverted terminal repeat, an AAV1 3’ inverted terminal repeat, an AAV2 3’ inverted terminal repeat, an AAV9 3’ inverted terminal repeat, or a PhP.eB 3’ inverted terminal repeat. In some aspects, the 5’ inverted terminal repeat is an AAV2 3 ’ inverted terminal repeat. In some aspects, the 3 ’ inverted terminal repeat comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 60.

[0014] In some aspects, the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 3; (ii) SEQ ID NO 10; (iii) SEQ ID NO: 8; (iv) SEQ ID NO: 42; and (v) SEQ ID NO: 64. In some aspects, the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 59; (ii) SEQ ID NO: 3; (iii) SEQ ID NO 10; (iv) SEQ ID NO: 8; (v) SEQ ID NO: 42; (vi) SEQ ID NO: 64; and (vii) SEQ ID NO: 60. In some aspects, the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 3; (ii) SEQ ID NO: 62, wherein the 3’ 33 nucleotides of SEQ ID NO: 3 overlap with the 5’ 33 nucleotides of SEQ ID NO: 62; (iii) SEQ ID NO 10; (iv) SEQ ID NO: 8; (v) SEQ ID NO: 42; and (vi) SEQ ID NO: 64. In some aspects, the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 3; (ii) SEQ ID NO: 66; (iii) SEQ ID NO 10; (iv) SEQ ID NO: 8; (v) SEQ ID NO: 42; and (vi) SEQ ID NO: 64. In some aspects, the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 3; (ii) SEQ ID NO: 56;

(iii) SEQ ID NO 10; (iv) SEQ ID NO: 8; (v) SEQ ID NO: 42; and (vi) SEQ ID NO: 64. In some aspects, the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 61 (ii) SEQ ID NO: 3; (iii) SEQ ID NO: 62, wherein the 3’ 33 nucleotides of SEQ ID NO: 3 overlap with the 5’ 33 nucleotides of SEQ ID NO: 62; (iv) SEQ ID NO 10; (v) SEQ ID NO: 8; (vi) SEQ ID NO: 42; and (vii) SEQ ID NO: 64. In some aspects, the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 61 (ii) SEQ ID NO: 3; (iii) SEQ ID NO: 66; (iv) SEQ ID NO 10; (v) SEQ ID NO: 8; (vi) SEQ ID NO: 42; and (vii) SEQ ID NO: 64. In some aspects, the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 61 (ii) SEQ ID NO: 3; (iii) SEQ ID NO: 56; (iv) SEQ ID NO 10; (v) SEQ ID NO: 8; (vi) SEQ ID NO: 42; and (vii) SEQ ID NO: 64. In some aspects, the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 59; (ii) SEQ ID NO: 3; (iii) SEQ ID NO: 62, wherein the 3’ 33 nucleotides of SEQ ID NO: 3 overlap with the 5’ 33 nucleotides of SEQ ID NO: 62; (iv) SEQ ID NO 10; (v) SEQ ID NO: 8; (vi) SEQ ID NO: 42; (vii) SEQ ID NO: 64; and (viii) SEQ ID NO: 60. In some aspects, the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 59; (ii) SEQ ID NO: 3; (iii) SEQ ID NO: 66; (iv) SEQ ID NO 10; (v) SEQ ID NO: 8; (vi) SEQ ID NO: 42; (vii) SEQ ID NO: 64; and (viii) SEQ ID NO: 60. In some aspects, the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 59; (ii) SEQ ID NO: 3; (iii) SEQ ID NO: 56; (iv) SEQ ID NO 10; (v) SEQ ID NO: 8; (vi) SEQ ID NO: 42; (vii) SEQ ID NO: 64; and (viii) SEQ ID NO: 60. In some aspects, the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 59; (ii) SEQ ID NO: 61; (iii) SEQ ID NO: 3; (iv) SEQ ID NO: 62, wherein the 3’ 33 nucleotides of SEQ ID NO: 3 overlap with the 5’ 33 nucleotides of SEQ ID NO: 62; (v) SEQ ID NO 10; (vi) SEQ ID NO: 8; (vii) SEQ ID NO: 42; (viii) SEQ ID NO: 64; and (ix) SEQ ID NO: 60. In some aspects, the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 59; (ii) SEQ ID NO: 61; (iii) SEQ ID NO: 3; (iv) SEQ ID NO: 66; (v) SEQ ID NO 10; (vi) SEQ ID NO: 8; (vii) SEQ ID NO: 42; (viii) SEQ ID NO: 64; and (ix) SEQ ID NO: 60. In some aspects, the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 59; (ii) SEQ ID NO: 61; (iii) SEQ ID NO: 3; (iv) SEQ ID NO: 56; (v) SEQ ID NO 10; (vi) SEQ ID NO: 8; (vii) SEQ ID NO: 42; (viii) SEQ ID NO: 64; and (ix) SEQ ID NO: 60. [0015] In some aspects, the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 65. In some aspects, the recombinant polynucleotide comprises a sequence of SEQ ID NO: 65. In some aspects, the recombinant polynucleotide is SEQ ID NO: 65. In some aspects, the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 121. In some aspects, the recombinant polynucleotide comprises a sequence of SEQ ID NO: 121. In some aspects, the recombinant polynucleotide is SEQ ID NO: 121. In some aspects, the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 122. In some aspects, the recombinant polynucleotide comprises a sequence of SEQ ID NO: 122. In some aspects, the recombinant polynucleotide is SEQ ID NO: 122. In some aspects, the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 123. In some aspects, the recombinant polynucleotide comprises a sequence of SEQ ID NO: 123. In some aspects, the recombinant polynucleotide is SEQ ID NO: 123. In some aspects, the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 124. In some aspects, the recombinant polynucleotide comprises a sequence of SEQ ID NO: 124. In some aspects, the recombinant polynucleotide is SEQ ID NO: 124.

[0016] In some aspects, the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 65.

[0017] In various aspects, the present disclosure provides a recombinant polynucleotide comprising: an RSV promoter; a coding sequence operably linked to the RSV promoter, wherein the coding sequence encodes a progranulin; and a post-transcriptional regulatory element.

[0018] In some aspects, the post-transcriptional regulatory element comprises a WPRE. In some aspects, the WPRE is a shortened WPRE. In some aspects, the shortened WPRE is a WPRE3 comprising at least at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 8. In some aspects, the WPRE3 comprises at least 90% sequence identity to SEQ ID NO: 8. In some aspects, the WPRE3 comprises a sequence of SEQ ID NO: 8. In some aspects, the WPRE comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 7. In some aspects, the WPRE comprises at least 90% sequence identity to SEQ ID NO: 7. In some aspects, the WPRE comprises a sequence of SEQ ID NO: 7. In some aspects, the post- transcriptional regulatory element is downstream of the coding sequence.

[0019] In various aspects, the present disclosure provides a recombinant polynucleotide comprising: an RSV promoter; a coding sequence operably linked to the RSV promoter, wherein the coding sequence encodes a progranulin; and a polyadenylation signal.

[0020] In some aspects, the polyadenylation signal comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 42, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 43 - SEQ ID NO: 48, or SEQ ID NO: 55. In some aspects, the polyadenylation signal comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 42. In some aspects, the polyadenylation signal comprises at least 90% sequence identity to SEQ ID NO: 42. In some aspects, the polyadenylation signal comprises a sequence of SEQ ID NO: 42. In some aspects, the polyadenylation signal is downstream of the coding sequence.

[0021] In various aspects, the present disclosure provides a recombinant polynucleotide comprising: an RSV promoter; a coding sequence operably linked to the RSV promoter, wherein the coding sequence encodes a progranulin; and a transcriptional pause site.

[0022] In some aspects, the transcriptional pause site comprises a second polyadenylation signal. In some aspects, the transcriptional pause site comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 64. In some aspects, the transcriptional pause site comprises at least 90% sequence identity to SEQ ID NO: 64. In some aspects, the transcriptional pause site comprises a sequence of SEQ ID NO: 64. In some aspects, the transcriptional pause site is downstream of the coding sequence. [0023] In some aspects, the coding sequence is downstream of the RSV promoter, the post- transcriptional regulatory element is downstream of the coding sequence, the polyadenylation signal is downstream of the post-transcriptional regulatory element, and the transcriptional pause site is downstream of the post-transcriptional regulatory element.

[0024] In some aspects, the RSV promoter comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 3.

[0025] In some aspects, the progranulin is a human progranulin. In some aspects, the progranulin encoded by the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 63. In some aspects, the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 6. In some aspects, the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 10. In some aspects, the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 11. In some aspects, the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 26. In some aspects, the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 57 and at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 58.

[0026] In some aspects, the recombinant polynucleotide further comprises a stuffer sequence. In some aspects, the stuffer sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 61. In some aspects, the stuffer sequence is upstream of the coding sequence. In some aspects, the stuffer sequence is upstream of the RSV promoter.

[0027] In some aspects, the recombinant polynucleotide comprises a 5’ untranslated region. In some aspects, the 5’ untranslated region comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 62, SEQ ID NO: 56, or SEQ ID NO: 66. In some aspects, the 5’ untranslated region is upstream of the coding sequence. In some aspects, the 5’ untranslated region is downstream of the RSV promoter.

[0028] In some aspects, the recombinant polynucleotide comprises a 5’ inverted terminal repeat upstream of the RSV promoter and a 3’ inverted terminal repeat downstream of the transcriptional pause site. In some aspects, the 5’ inverted terminal repeat is an AAV 5’ inverted terminal repeat. In some aspects, the 5’ inverted terminal repeat is an AAV5 5’ inverted terminal repeat, an AAV1 5’ inverted terminal repeat, an AAV2 5’ inverted terminal repeat, an AAV9 5’ inverted terminal repeat, or a PhP.eB 5’ inverted terminal repeat. In some aspects, the 5’ inverted terminal repeat comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 59. In some aspects, the 3’ inverted terminal repeat is an AAV 5’ inverted terminal repeat. In some aspects, the 3’ inverted terminal repeat is an AAV5 3’ inverted terminal repeat, an AAV1 3’ inverted terminal repeat, an AAV2 3’ inverted terminal repeat, an AAV9 3’ inverted terminal repeat, or a PhP.eB 3’ inverted terminal repeat. In some aspects, the 3’ inverted terminal repeat comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 60. [0029] In some aspects, the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 65.

[0030] In some aspects, the recombinant polynucleotide further comprises a neuron-restrictive silencer element. In some aspects, the neuron-restrictive silencer element comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to any one of SEQ ID NO: 32 - SEQ ID NO: 39.

[0031] In some aspects, the recombinant polynucleotide comprises a CCCTC-binding factor sequence. In some aspects, the CCCTC-binding factor sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 38.

[0032] In some aspects, the recombinant polynucleotide further comprises an intron. In some aspects, the intron is within a 5’ untranslated region of the recombinant polynucleotide. In some aspects, the intron is within the coding sequence. In some aspects, the intron comprises a GRN intron, an SV40 intron, a TPI intron, or a combination thereof. In some aspects, the GRN intron comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 12. In some aspects, the GRN intron comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity any one of SEQ ID NO: 27 - SEQ ID NO: 29. In some aspects, the SV40 intron comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 13. In some aspects, the SV40 intron comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 30. In some aspects, the TPI intron comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 31.

[0033] In some aspects, the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO:

67. In some aspects, the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO:

68. In some aspects, the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO:

69. In some aspects, the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO:

70. In some aspects, the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO:

15. In some aspects, the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO:

16. In some aspects, the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO:

17. In some aspects, the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO:

18.

[0034] In various aspects, the present disclosure provides a plasmid comprising a recombinant polynucleotide as described herein.

[0035] In some aspects, the plasmid comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 120. In some aspects, the plasmid is SEQ ID NO: 120. In some aspects, the plasmid comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 19. In some aspects, the plasmid comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 20. In some aspects, the plasmid comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 50. In some aspects, the plasmid comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 53. In some aspects, the plasmid comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 71. In some aspects, the plasmid comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 73. In some aspects, the plasmid comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 74.

[0036] In various aspects, the present disclosure provides a viral vector comprising a recombinant polynucleotide as described herein or a plasmid as described herein.

[0037] In some aspects, the viral vector is an adenoviral vector, an adeno-associated viral (AAV) vector, or a lentivector. In some aspects, the adeno-associated viral vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-DJ/8, AAV-DJ/9, AAV1/2, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAV.OligoOOl, AAV.SCH9, AAV.r3.45, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PhP.eB, AAV.PhP.Vl, AAV.PHP.B, AAV.PhB.Cl, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, AAVhu68, chimeras thereof, and combinations thereof. In some aspects, the AAV vector is an AAV5 vector. In some aspects, the AAV vector comprises an engineered viral protein (VP) capsid polypeptide. In some aspects, the engineered VP capsid polypeptide comprises at least one substitution in a 581-589 region of the engineered VP capsid polypeptide relative to a wild type VP capsid polypeptide of SEQ ID NO: 75. In some aspects, the 581-589 region comprises a sequence of SEQ ID NO: 83. In some aspects, the 581-589 region comprises a sequence of SEQ ID NO: 84. In some aspects, the 581-589 region comprises a sequence of SEQ ID NO: 96. In some aspects, the 581-589 region comprises a sequence of SEQ ID NO: 82. In some aspects, the 581-589 region comprises a sequence of any one of SEQ ID NO: 84 - SEQ ID NO: 95 or SEQ ID NO: 97 - SEQ ID NO: 111. In some aspects, the engineered VP capsid polypeptide comprises a formula of (A)-(X)-(B), wherein (A) is a first polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 80, (X) is the 581-589 region, and (B) is a second polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 81.

[0038] In various aspects, the present disclosure provides a pharmaceutical composition comprising a recombinant polynucleotide as described herein, a plasmid as described herein, or a viral vector as described herein, and a pharmaceutically acceptable excipient, carrier, diluent, or combination thereof.

[0039] In various aspects, the present disclosure provides a method of expressing a progranulin in a cell, tissue, or subject, the method comprising: delivering a recombinant polynucleotide comprising an RSV promoter and a coding sequence operably linked to the RSV promoter, to the cell, tissue, or subject; and expressing a progranulin encoded by the coding sequence.

[0040] In various aspects, the present disclosure provides a method of expressing a progranulin in a cell, tissue, or subject, the method comprising: delivering a recombinant polynucleotide as described herein, a plasmid as described herein, a viral vector as described herein, or a pharmaceutical composition as described herein to the cell, tissue, or subject.

[0041] In some aspects, the progranulin is human progranulin. In some aspects, the progranulin encoded by the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 63. In some aspects, the progranulin is encoded by a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 10. In some aspects, the progranulin is encoded by a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 6. In some aspects, the progranulin is encoded by a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 11. In some aspects, the progranulin is encoded by a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 26. In some aspects, the progranulin is encoded by a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 57 and at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 58.

[0042] In some aspects, the method comprises modulating expression of the progranulin in the cell, tissue, or subject with a neuron-restrictive silencer element.

[0043] In some aspects, the method comprises expressing the progranulin in a fluid secreted by the cell, tissue, or subject. In some aspects, the progranulin is expressed in the fluid at a level of not less than 1 ng/mL and not more than 100 ng/mL. In some aspects, the progranulin is expressed in the fluid at a level of not less than 10 ng/mL and not more than 100 ng/mL. In some aspects, the progranulin is expressed in the fluid at a level of not less than 1 ng/mL and not more than 30 ng/mL. In some aspects, the progranulin is expressed in the fluid at a level of not less than 10 ng/mL and not more than 50 ng/mL. In some aspects, the progranulin is expressed in the fluid at a level of not less than 10 ng/mL and not more than 30 ng/mL. In some aspects, the progranulin is expressed in the fluid at a level of not less than 70 ng/mL and not more than 180 ng/mL. In some aspects, the progranulin is expressed in the fluid at a level of not less than 70 ng/mL and not more than 300 ng/mL. In some aspects, the fluid comprises a cerebrospinal fluid. [0044] In various aspects, the present disclosure provides a method of treating a disease or disorder in a subject in need thereof, the method comprising: delivering a recombinant polynucleotide comprising an RSV promoter and a coding sequence operably linked to the RSV promoter to the subject having frontotemporal dementia; and expressing a progranulin encoded by the coding sequence, thereby treating the subject.

[0045] In various aspects, the present disclosure provides a method of treating a disease or disorder in a subject in need thereof, the method comprising: delivering a recombinant polynucleotide as described herein, a plasmid as described herein, a viral vector as described herein, or a pharmaceutical composition as described herein to the subject in need thereof, thereby treating the disease or disorder. [0046] In some aspects, the disease or disorder is frontotemporal dementia, amyotrophic lateral sclerosis (ALS), Alzheimer’s Disease, Parkinson’s Disease, stroke, Gaucher disease, arthritis, limbic-predominant age-related transactivation response DNA-binding protein 43 (TDP-43) encephalopathy, autism, neuronal ceroid lipofuscinosis (e.g., type 11 (CLN11)), dementia, and neurodegeneration; optionally, wherein the neuronal ceroid lipofuscinosis is type 11 ; optionally, wherein neurodegeneration is neurodegeneration associated with normal aging.

[0047] In various aspects, the present disclosure provides a method of treating frontotemporal dementia in a subject having frontotemporal dementia, the method comprising: delivering a recombinant polynucleotide comprising an RSV promoter and a coding sequence operably linked to the RSV promoter to the subject having frontotemporal dementia; and expressing a progranulin encoded by the coding sequence, thereby treating the subject.

[0048] In various aspects, the present disclosure provides a method of treating frontotemporal dementia in a subject having frontotemporal dementia, the method comprising: delivering a recombinant polynucleotide as described herein, a plasmid as described herein, a viral vector as described herein, or a pharmaceutical composition as described herein to the subject having frontotemporal dementia, thereby treating the frontotemporal dementia.

[0049] In some aspects, the method comprises expressing the progranulin in a cerebrospinal fluid of the subject. In some aspects, the progranulin is expressed in the cerebrospinal fluid at a level of not less than 10 ng/mL and not more than 100 ng/mL. In some aspects, the progranulin is expressed in the cerebrospinal fluid at a level of not less than 1 ng/mL and not more than 100 ng/mL. In some aspects, the progranulin is expressed in the cerebrospinal fluid at a level of not less than 10 ng/mL and not more than 30 ng/mL. In some aspects, the progranulin is expressed in the cerebrospinal fluid at a level of not less than 1 ng/mL and not more than 30 ng/mL. In some aspects, the progranulin is expressed in the cerebrospinal fluid at a level of not less than 10 ng/mL and not more than 50 ng/mL. In some aspects, the progranulin is expressed in the cerebrospinal fluid at a level of not less than 10 ng/mL and not more than 30 ng/mL. In some aspects, the progranulin is expressed in the cerebrospinal fluid at a level of not less than 70 ng/mL and not more than 180 ng/mL. In some aspects, the progranulin is expressed in the cerebrospinal fluid at a level of not less than 70 ng/mL and not more than 300 ng/mL.

[0050] In some aspects, an expression level of the progranulin in the cerebrospinal fluid is not more than 100-fold, not more than 50-fold, not more than 25 -fold, not more than 20-fold, or not more than 10-fold an expression level of the progranulin in a serum of the subject. In some aspects, an expression level of the progranulin in the cerebrospinal fluid is not less than 0.01- fold, not less than 0.05 -fold, not less than 0.1 -fold, not less than 0.2-fold, not less than 0.25 -fold, or not less than 0.5-fold an expression level of the progranulin in a serum of the subject. In some aspects, an expression level of the progranulin in a serum of the subject is not more than 100- fold, not more than 50-fold, not more than 25-fold, not more than 20-fold, or not more than 10- fold an expression level of the progranulin in the cerebrospinal fluid. In some aspects, an expression level of the progranulin in a serum of the subject is not less than 0.01 -fold, not less than 0.05-fold, not less than 0.1 -fold, not less than 0.2-fold, not less than 0.25-fold, or not less than 0.5-fold an expression level of the progranulin in the cerebrospinal fluid. In some aspects, an expression level of the progranulin in the cerebrospinal fluid higher than an expression level of the progranulin in a serum of the subject. In some aspects, an expression level of the progranulin in the cerebrospinal fluid is not less than 0.1 -fold and not more than 100-fold an expression level of the progranulin in a serum of the subject. In some aspects, an expression level of the progranulin in a serum of the subject is not more than 300 ng/mL. In some aspects, an expression level of the progranulin in a serum of the subject is not more than 1500 ng/mL. [0051] In some aspects, the subject is a mammal. In some aspects, the mammal is a human, a non-human primate, a rat, a rabbit, a mouse, or a guinea pig. In some aspects, the delivering comprises intravenous administration.

[0052] In various aspects, the present disclosure provides a recombinant polynucleotide comprising a promoter and a sequence encoding a payload, wherein the promoter is operably linked to the sequence encoding the payload; wherein the promoter is selected from a group consisting of a CAG promoter, a CMV promoter, an RSV promoter, a JeT promoter, a synapsin promoter, a minimal CMV promoter, an EF- la promoter, a putative MECP2 promoter, a B-actin promoter, a MND promoter, and a ybTATA promoter; and wherein the payload is a progranulin. [0053] In some aspects, the payload is a human progranulin. In some aspects, the sequence encoding the payload is a GRN sequence. In some aspects, the sequence encoding the payload is a human GRN sequence. In some aspects, the sequence encoding the payload comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 6. In some aspects, the sequence encoding the payload comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 10. In some aspects, the sequence encoding the payload comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 11.

[0054] In some aspects, the promoter is selected from the group consisting of a CAG promoter, an RSV promoter, a JeT promoter, and a synapsin promoter. In some aspects, the promoter is the CAG promoter. In some aspects, the CAG promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 1. In some aspects, the promoter is the CMV promoter. In some aspects, the CMV promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 2. In some aspects, the promoter is the RSV promoter. In some aspects, the RSV promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 3. In some aspects, the promoter is the JeT promoter. In some aspects, the JeT promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 4. In some aspects, the promoter is the Synapsin promoter. In some aspects, the Synapsin promoter is a human Synapsin (hSynapsin) promoter. In some aspects, the synapsin promoter comprises at 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 5. In some aspects, the promoter is the minimal CMV promoter. In some aspects, the minimal CMV promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 21. In some aspects, the promoter is the EF-la promoter. In some aspects, the EF-la promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 22. In some aspects, the promoter is the putative MECP2 promoter. In some aspects, the putative MECP2 promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 23. In some aspects, the promoter is the B-actin promoter. In some aspects, the B-actin promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 24. In some aspects, the promoter is the MND promoter. In some aspects, the MND promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 21. In some aspects, the promoter is the ybTATA promoter. In some aspects, the ybTATA promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 54.

[0055] In some aspects, the recombinant polynucleotide further comprises an additional sequence element. In some aspects, the additional sequence element comprises a WPRE element, a neuron-restrictive silencer element, a WPRE3 element, a neuron-restrictive silencer element, a CCCTC-binding factor sequence, a polyadenylation signal, a 5’ untranslated region, a 3 ’ untranslated region, or combinations thereof. In some aspects, the additional sequence element comprises the WPRE element. In some aspects, the WPRE element comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 7. In some aspects, the additional sequence element comprises the WPRE3 element. In some aspects, the WPRE3 element comprises at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 8. In some aspects, the additional sequence element comprises the neuron- restrictive silencer element. In some aspects, the neuron-restrictive silencer element comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to any one of SEQ ID NO: 32 - SEQ ID NO: 39. In some aspects, the additional sequence element comprises the CCCTC- binding factor sequence. In some aspects, the CCCTC-binding factor sequence comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 30. In some aspects, the additional sequence element comprises the polyadenylation signal downstream of the sequence encoding the payload. In some aspects, the polyadenylation signal comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to any one of SEQ ID NO: 40 - SEQ ID NO: 48. In some aspects, the polyadenylation signal comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 42. In some aspects, the additional sequence element comprises the 5’ untranslated region upstream of the sequence encoding the payload. In some aspects, the additional sequence element comprises the 3 ’ untranslated region downstream of the sequence encoding the payload. In some aspects, the 3’ untranslated region comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 40 or SEQ ID NO: 41. [0056] In some aspects, the recombinant polynucleotide further comprises an intron. In some aspects, the intron is within a 5’ untranslated region of the recombinant polynucleotide. In some aspects, the intron is within the sequence encoding the payload. In some aspects, the intron comprises a GRN intron, an SV40 intron, a TPI intron, or a combination thereof. In some aspects, the GRN intron comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 12. In some aspects, the GRN intron comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity any one of SEQ ID NO: 27 - SEQ ID NO: 29. In some aspects, the SV40 intron comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 13. In some aspects, the SV40 intron comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 30. In some aspects, the TPI intron comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 31.

[0057] In some aspects, the recombinant polynucleotide comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 14. In some aspects, the recombinant polynucleotide comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 15. In some aspects, the recombinant polynucleotide comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 16. In some aspects, the recombinant polynucleotide comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 17. In some aspects, the recombinant polynucleotide comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 18. [0058] In various aspects, the present disclosure provides a recombinant polynucleotide comprising a promoter and a sequence encoding a payload, wherein the promoter is operably linked to the sequence encoding the payload, wherein the sequence encoding the payload comprises an intron, wherein the payload is progranulin, and wherein the intron is a GRN native intron 9.

[0059] In some aspects, the GRN native intron 9 comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 12. In some aspects, the progranulin is a human progranulin. In some aspects, the sequence encoding the payload comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 11. In some aspects, the recombinant polynucleotide further comprises a 5 ’ untranslated region upstream of the sequence encoding the payload. In some aspects, the recombinant polynucleotide further comprises a second intron within the 5’ untranslated region. In some aspects, the second intron is an SV40 intron. In some aspects, the SV40 intron comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 13.

[0060] In various aspects, the present disclosure provides a recombinant polynucleotide comprising a promoter, a sequence encoding a payload, and a 5 ’ untranslated region upstream of the sequence encoding the payload, wherein the promoter is operably linked to the sequence encoding the payload, wherein the payload is progranulin, wherein the 5’ untranslated region comprises an intron, and wherein the intron is an SV40 intron.

[0061] In some aspects, the SV40 intron comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 13. In some aspects, the progranulin is a human progranulin. In some aspects, the sequence encoding the payload comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 11. In some aspects, the sequence encoding the payload comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 6. In some aspects, the sequence encoding the payload comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 10. In some aspects, wherein the recombinant polynucleotide comprises a second intron within the sequence encoding the payload. In some aspects, the second intron is a GRN intron 9. In some aspects, the GRN intron 9 comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 12.

[0062] In some aspects, the promoter is selected from a group consisting of a CAG promoter, a CMV promoter, an RSV promoter, a JeT promoter, a synapsin promoter, a minimal CMV promoter, an EF-la promoter, a putative MECP2 promoter, a B-actin promoter, a MND promoter, and a ybTATA promoter. In some aspects, the promoter is selected from the group consisting of a CAG promoter, an RSV promoter, a JeT promoter, and a synapsin promoter. In some aspects, the promoter is the CAG promoter. In some aspects, the CAG promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 1. In some aspects, the promoter is the CMV promoter. In some aspects, the CMV promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 2. In some aspects, the promoter is the RSV promoter. In some aspects, the RSV promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 3. In some aspects, the promoter is the JeT promoter. In some aspects, the JeT promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 4. In some aspects, the promoter is the Synapsin promoter. In some aspects, the Synapsin promoter is a human Synapsin (hSynapsin) promoter. In some aspects, the synapsin promoter comprises at 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 5. In some aspects, the promoter is the minimal CMV promoter. In some aspects, the minimal CMV promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO:

21. In some aspects, the promoter is the EF-la promoter. In some aspects, the EF-la promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO:

22. In some aspects, the promoter is the putative MECP2 promoter. In some aspects, the putative MECP2 promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 23. In some aspects, the promoter is the B-actin promoter. In some aspects, the B-actin promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 24. In some aspects, the promoter is the MND promoter. In some aspects, the MND promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 21. In some aspects, the promoter is the ybTATA promoter. In some aspects, the ybTATA promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 54.

[0063] In some aspects, the recombinant polynucleotide further comprises an additional sequence element. In some aspects, the additional sequence element comprises a WPRE element, a neuron-restrictive silencer element, a WPRE3 element, a neuron-restrictive silencer element, a CCCTC-binding factor sequence, a polyadenylation signal, a 5’ untranslated region, a 3 ’ untranslated region, or combinations thereof. In some aspects, the additional sequence element comprises the WPRE element. In some aspects, the WPRE element comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 7. In some aspects, the additional sequence element comprises the WPRE3 element. In some aspects, the WPRE3 element comprises at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 8. In some aspects, the additional sequence element comprises the neuron- restrictive silencer element. In some aspects, the neuron-restrictive silencer element comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to any one of SEQ ID NO: 32 - SEQ ID NO: 39. In some aspects, the additional sequence element comprises the CCCTC- binding factor sequence. In some aspects, the CCCTC-binding factor sequence comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 30. In some aspects, the additional sequence element comprises the polyadenylation signal downstream of the sequence encoding the payload. In some aspects, the polyadenylation signal comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to any one of SEQ ID NO: 40 - SEQ ID NO: 48. In some aspects, the polyadenylation signal comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 42. In some aspects, the additional sequence element comprises the 5’ untranslated region upstream of the sequence encoding the payload. In some aspects, the additional sequence element comprises the 3 ’ untranslated region downstream of the sequence encoding the payload. In some aspects, the 3’ untranslated region comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 40 or SEQ ID NO: 41.

[0064] In various aspects, the present disclosure provides a plasmid comprising a recombinant polynucleotide as described herein.

[0065] In some aspects, the recombinant polynucleotide is flanked by inverted terminal repeats. In some aspects, the plasmid comprises at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 9. In some aspects, the plasmid comprises at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 19. In some aspects, the plasmid comprises at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 20. In some aspects, the plasmid comprises at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 49. In some aspects, the plasmid comprises at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 50. In some aspects, the plasmid comprises at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 51. In some aspects, the plasmid comprises at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 52. In some aspects, the plasmid comprises at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 53.

[0066] In various aspects, the present disclosure provides a viral vector encapsidating a recombinant polynucleotide as described herein. [0067] In some aspects, the plasmid comprises the recombinant polynucleotide comprises at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to the sequence of the recombinant polynucleotide between inverted terminal repeats in SEQ ID NO: 9. In some aspects, the plasmid comprises the viral vector is an adenoviral vector, an adeno-associated viral vector, or a lentivector. In some aspects, the plasmid comprises the adeno-associated viral vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-DJ/8, AAV-DJ/9, AAV1/2, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAV.OligoOOl, AAV.SCH9, AAV.r3.45, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PhP.eB, AAV.PhP.Vl, AAV.PHP.B, AAV.PhB.Cl, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, AAVhu68, chimeras thereof, and combinations thereof.

[0068] In various aspects, the present disclosure provides a pharmaceutical composition comprising a recombinant polynucleotide as described herein, a plasmid as described herein, or a viral vector as described herein, and a pharmaceutically acceptable excipient, carrier, diluent, or combination thereof.

[0069] In various aspects, the present disclosure provides a method of expressing a payload in a cell, tissue, or subject, the method comprising: delivering a recombinant polynucleotide as described herein, a plasmid as described herein, a viral vector as described herein, or a pharmaceutical composition as described herein to the cell, tissue, or subject.

[0070] In some aspects, the payload is a progranulin. In some aspects, the progranulin is human progranulin. In some aspects, the progranulin comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 6. In some aspects, the progranulin comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 10. In some aspects, the progranulin comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 11.

[0071] In some aspects, the method comprises modulating expression of the payload in the cell, tissue, or subject with a tissue-specific promoter or a cell-specific promoter. In some aspects, the method comprises modulating expression of the payload in the cell, tissue, or subject with a neuron-restrictive silencer element. [0072] In various aspects, the present disclosure provides a method of expressing a progranulin in a cell, tissue, or subject, the method comprising: delivering a recombinant polynucleotide as described herein, a plasmid as described herein, a viral vector as described herein, or a pharmaceutical composition as described herein to the cell, tissue, or subject.

[0073] In some aspects, the method comprises modulating expression of the progranulin in the cell, tissue, or subject with a tissue-specific promoter or a cell-specific promoter. In some aspects, the method comprises modulating expression of the progranulin in the cell, tissue, or subject with a neuron-restrictive silencer element.

[0074] In various aspects, the present disclosure provides a method of treating a disease or disorder in a subject in need thereof, the method comprising: delivering a recombinant polynucleotide as described herein, a plasmid as described herein, a viral vector as described herein, or a pharmaceutical composition as described herein to the subject in need thereof, thereby treating the disease or disorder.

[0075] In some aspects, the disease or disorder is frontotemporal dementia, amyotrophic lateral sclerosis (ALS), Alzheimer’s Disease, Parkinson’s Disease, stroke, Gaucher disease, arthritis, limbic-predominant age-related transactivation response DNA-binding protein 43 (TDP-43) encephalopathy, autism, neuronal ceroid lipofuscinosis (e.g., type 11 (CLN11)), dementia, and neurodegeneration; optionally, wherein the neuronal ceroid lipofuscinosis is type 11 ; optionally, wherein neurodegeneration is neurodegeneration associated with normal aging.

[0076] In various aspects, the present disclosure provides a method of treating frontotemporal dementia in a subject having frontotemporal dementia, the method comprising: delivering a recombinant polynucleotide as described herein, a plasmid as described herein, a viral vector as described herein, or a pharmaceutical composition as described herein to the subject having frontotemporal dementia, thereby treating the frontotemporal dementia.

[0077] In some aspects, the method further comprises expressing a progranulin in a cerebrospinal fluid of the subject. In some aspects, an expression level of the progranulin in the cerebrospinal fluid is not less than 1 ng/mL and not more than 3 ng/mL, not less than 2 ng/mL and not more than 4.5 ng/mL, or not less than 1 ng/mL and not more than 10 ng/mL. In some aspects, an expression level of the progranulin in the cerebrospinal fluid is not less than 1 ng/mL and not more than 4 ng/mL. In some aspects, an expression level of the progranulin in the cerebrospinal fluid is not less than 3 ng/mL. In some aspects, an expression level of the progranulin in the cerebrospinal fluid is not more than 100-fold, not more than 50-fold, not more than 25-fold, not more than 20-fold, or not more than 10-fold an expression level of the progranulin in a serum of the subject. In some aspects, an expression level of the progranulin in the cerebrospinal fluid is not less than 0.01 -fold, not less than 0.05-fold, not less than 0.1 -fold, not less than 0.2-fold, not less than 0.25 -fold, or not less than 0.5 -fold an expression level of the progranulin in a serum of the subject. In some aspects, an expression level of the progranulin in a serum of the subject is not more than 100-fold, not more than 50-fold, not more than 25 -fold, not more than 20-fold, or not more than 10-fold an expression level of the progranulin in the cerebrospinal fluid. In some aspects, an expression level of the progranulin in a serum of the subject is not less than 0.01-fold, not less than 0.05-fold, not less than 0.1-fold, not less than 0.2- fold, not less than 0.25-fold, or not less than 0.5-fold an expression level of the progranulin in the cerebrospinal fluid. In some aspects, an expression level of the progranulin in the cerebrospinal fluid higher than an expression level of the progranulin in a serum of the subject. In some aspects, an expression level of the progranulin in the cerebrospinal fluid is not less than 0.1 -fold and not more than 100-fold an expression level of the progranulin in a serum of the subject.

[0078] In some aspects, the subject is a mammal. In some aspects, the mammal is a human, a non-human primate, a rat, a rabbit, a mouse, or a guinea pig.

INCORPORATION BY REFERENCE

[0079] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0080] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0081] FIG. 1 shows a plasmid (SEQ ID NO: 9) comprising a recombinant polynucleotide having a CAG promoter (SEQ ID NO: 1) operably linked to a sequence encoding progranulin (SEQ ID NO: 6) and having a WPRE (SEQ ID NO: 7), a chimeric intron, and a rabbit beta globin polyA (SEQ ID NO: 42), flanked by ITRs for producing single stranded AAV (ssAAV). [0082] FIG. 2 shows that progranulin was detected in supernatant from HEK293 cells infected at either 6xl0⁴ vg/cell (6e4 vg/cell) or 2xl0⁴ vg/cell (2e4 vg/cell) with AAV9-CAG-GRN (AAV produced using a plasmid of SEQ ID NO: 9) compared to no progranulin detection in the cells not infected by AAV9 virus (no virus). [0083] FIG. 3 shows relative body weights of mice post-ICV injection of ssAAV9-CAG-GRN (“CAG-GRN”; top row) or scAAV9-CMV-GFP (“control”; bottom row) over a time period of 3 weeks, 4 weeks, or 6 weeks after ICV injection (from left to right).

[0084] FIG. 4 shows ELIS As of human progranulin in the CSF at 3, 4, and 6 weeks post-ICV injection of ssAAV9-CAG-GRN compared to the control (scAAV9-CMV-GFP) at 3, 4 and 6 weeks post-ICV injection. Asterisks denote statistical significance, as determined by Tukey’s Honest Significant Difference (HSD) test (*** P<0.003; ** P<0.01; * P<0.05).

[0085] FIG. 5 shows ELIS As of human progranulin (hGRN) in the serum at 2, 3, 4, and 6 weeks post-ICV injection of ssAAV9-CAG-GRN compared to the control (scAAV9-CMV- GFP) at 3, 4 and 6 weeks post-ICV injection. Asterisks denote statistical significance, as determined by Tukey’s HSD test (**** P0.001; * P<0.05).

[0086] FIG. 6 shows a correlation graph of the CSF and serum for detected progranulin (GRN).

[0087] FIG. 7A shows relative body weights of mice post-ICV injection of scAAV9- RSV hGRN (AAV produced using a plasmid of SEQ ID NO: 50; top left), scAAV9- JET hGRN (AAV produced using a plasmid of SEQ ID NO: 49; top right), ssAAV9-RSV- hGRN (AAV produced using a plasmid of SEQ ID NO: 53; bottom left), or ssAAV9- CAG_hGRN_WPRE (AAV produced using a plasmid of SEQ ID NO: 9; bottom right).

[0088] FIG. 7B shows relative body weights of mice post-ICV injection of scAAV9- RSV_hGRN_SV40int99 (AAV produced using a plasmid of SEQ ID NO: 19; top left), scAAV9-RSV_hGRN_int9 (AAV produced using a plasmid of SEQ ID NO: 20; top right), ssAAV9-Syn_hGRN_WPRE (AAV produced using a plasmid of SEQ ID NO: 52; bottom left), ssAAV9-CMV_hGRN_WPRE (AAV produced using a plasmid of SEQ ID NO: 51; bottom right).

[0089] FIG. 8 A and FIG. 8B shows ELIS As of human progranulin (GRN protein) in the CSF following ICV injection of scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6) (“scAAV9-RSV_hGRN”; AAV produced using a plasmid of SEQ ID NO: 50), scAAV9 encapsidating a JeT promoter (SEQ ID NO: 4) operably linked to GRN coding sequence (SEQ ID NO: 6) (“scAAV9-JET_hGRN”; AAV produced using a plasmid of SEQ ID NO: 49), scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6) and SV40 intron (SEQ ID NO: 13) (“scAAV9-RSV_hGRN_SV40int99;” AAV produced using a plasmid of SEQ ID NO: 19), scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence containing GRN intron 9 (SEQ ID NO: 11) (“scAAV9-RSV_hGRN_int9;” AAV produced using a plasmid of SEQ ID NO: 20), ssAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6) (“ssAAV9-RSV-hGRN;” AAV produced using a plasmid of SEQ ID NO: 53), ssAAV9 encapsidating a CAG promoter (SEQ ID NO: 1) operably linked to GRN coding sequence (SEQ ID NO: 6) and WPRE sequence (SEQ ID NO: 7) (“ssAAV9-CAG_hGRN_WPRE;” AAV produced using a plasmid of SEQ ID NO: 9), ssAAV9 encapsidating a hSyn promoter (SEQ ID NO: 5) operably linked to GRN coding sequence (SEQ ID NO: 6) and WPRE sequence (SEQ ID NO: 7) (“ssAAV9- Syn_hGRN_WPRE;” AAV produced using a plasmid of SEQ ID NO: 52), or ssAAV9 encapsidating a CMV promoter (SEQ ID NO: 2) operably linked to GRN coding sequence (SEQ ID NO: 6) and WPRE sequence (SEQ ID NO: 7) (“ssAAV9-CMV_hGRN_WPRE;” AAV produced using a plasmid of SEQ ID NO: 51). In FIG. 8A, asterisks denote statistical significance, as determined by one-way ANOVA and Dunnett’s multiple comparison test (**** P<0.0001; *** P<0.003; * P<0.05). In FIG. 8B, asterisks denote statistical significance, as determined by one-way ANOVA and Tukey’s multiple comparison test (**** P<0.0001; *** P<0.003; ** P<0.001; * P<0.05).

[0090] FIG. 9 shows ELIS As of human progranulin in the CSF following ICV injection of ssAAV9-Syn_hGRN_WPRE (AAV produced using a plasmid of SEQ ID NO: 52). The dotted line denotes a target therapeutic level of 3 ng/mL progranulin in the CSF. Statistics were performed using one-way ANOVA and Dunnett’s multiple comparison test (ns: not significant). [0091] FIG. 10 shows ELIS As of human progranulin in serum at 2 weeks post-ICV injection of scAAV9-RSV_hGRN (AAV produced using a plasmid of SEQ ID NO: 50), scAAV9- JET hGRN (AAV produced using a plasmid of SEQ ID NO: 49), scAAV9- RSV_hGRN_SV40int99 (AAV produced using a plasmid of SEQ ID NO: 19), scAAV9- RSV_hGRN_int9 (AAV produced using a plasmid of SEQ ID NO: 20), ssAAV9-RSV-hGRN (AAV produced using a plasmid of SEQ ID NO: 53), ssAAV9-CAG_hGRN_WPRE (AAV produced using a plasmid of SEQ ID NO: 9), ssAAV9-Syn_hGRN_WPRE (AAV produced using a plasmid of SEQ ID NO: 52), or ssAAV9-CMV_hGRN_WPRE (AAV produced using a plasmid of SEQ ID NO: 51). Asterisks denote statistical significance, as determined by Tukey’s HSD test (**** P0.0001; ** P0.001; * P<0.05).

[0092] FIG. 11 shows ELIS As of human progranulin in serum at 4 weeks post-ICV injection of scAAV9-RSV_hGRN (AAV produced using a plasmid of SEQ ID NO: 50), scAAV9- JET hGRN (AAV produced using a plasmid of SEQ ID NO: 49), scAAV9- RSV_hGRN_SV40int99 (AAV produced using a plasmid of SEQ ID NO: 19), scAAV9- RSV_hGRN_int9 (AAV produced using a plasmid of SEQ ID NO: 20), ssAAV9-RSV-hGRN (AAV produced using a plasmid of SEQ ID NO: 53), ssAAV9-CAG_hGRN_WPRE (AAV produced using a plasmid of SEQ ID NO: 9), ssAAV9-Syn_hGRN_WPRE (AAV produced using a plasmid of SEQ ID NO: 52), or ssAAV9-CMV_hGRN_WPRE (AAV produced using a plasmid of SEQ ID NO: 51). Asterisks denote statistical significance, as determined by Tukey’s HSD test (**** P<0.0001; ** P<0.001; * P<0.05).

[0093] FIG. 12A schematically illustrates a dual reporter plasmid containing a protein testing cassette and a transfection control cassette used in FIG. 12B and FIG. 12C. The protein testing cassette includes, from 5’ to 3’, a promoter, a 5’ untranslated region, an mCherry coding sequence, and a polyadenylation signal (“polyA sig”). The transfection control cassette includes, from 5’ to 3’, a CMV promoter (SEQ ID NO: 2), a GFP coding sequence, and a polyadenylation signal (“polyA sig”).

[0094] FIG. 12B shows mCherry to GFP fluorescence ratios in SH-SY5Y cells transfected with the dual reporter plasmid of FIG. 12A including a protein testing cassette containing either a EF1α promoter (SEQ ID NO: 22), a first synthetic promoter (Synth 1), a second synthetic promoter (Synth 2), a third synthetic promoter (Synth 3), a fourth synthetic promoter (Synth 4), a JeT promoter (SEQ ID NO: 4), an MECP2 promoter (SEQ ID NO: 23), an RSV promoter (SEQ ID NO: 3), a YB-TATA promoter (SEQ ID NO: 54), a chicken B-actin promoter (SEQ ID NO: 24), or an MND promoter (SEQ ID NO: 25). Cells transfected with a pMax vector were used as a negative control.

[0095] FIG. 12C shows mCherry to GFP fluorescence ratios in H4 cells transfected with the dual reporter plasmid of FIG. 12A including a protein testing cassette containing either a EF1α promoter (SEQ ID NO: 22), a JeT promoter (SEQ ID NO: 4), an MECP2 promoter (SEQ ID NO: 23), an RSV promoter (SEQ ID NO: 3), a chicken B-actin promoter (SEQ ID NO: 24), or an MND promoter (SEQ ID NO: 25). Cells transfected with a pMax vector were used as a negative control.

[0096] FIG. 13A schematically illustrates a reporter plasmid used in FIG. 13B. The reporter plasmid includes, from 5’ to 3’, a CMV promoter (SEQ ID NO: 2), a luciferase coding sequence (“Sec. Luciferase”), and a polyadenylation signal (“polyA sig”).

[0097] FIG. 13B shows luminescence data from HEK293 cells transfected with the reporter plasmid of FIG. 13A including a polyadenylation signal of SEQ ID NO: 45, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 46, SEQ ID NO: 40, or SEQ ID NO: 41. Cells transfected with a pMax vector and water only (“H2O”) were used as negative controls.

[0098] FIG. 14A schematically illustrates an expression plasmid containing a GRN expression cassette and a normalization expression cassette used in FIG. 14B. The GRN expression cassette includes, from 5’ to 3’, a promoter, a 5’ untranslated region, a progranulin coding sequence (“progranulin CDS”) containing an intron, and a polyadenylation signal (“polyA sig”). The normalization expression cassette includes, from 5’ to 3’, a CMV promoter (SEQ ID NO: 2), a luciferase coding sequence (“Sec. Luciferase”), and a polyadenylation signal (“polyA sig”). [0099] FIG. 14B shows ELISA data of progranulin expression normalized to luciferase activity from cells transfected with the expression plasmid of FIG. 14A including a GRN expression cassette containing a JeT promoter (SEQ ID NO: 4) and a human progranulin coding sequence (SEQ ID NO: 6) with no introns (“Jet”), an RSV promoter (SEQ ID NO: 3) and a human progranulin coding sequence (SEQ ID NO: 6) with no introns (“RSV”), a JeT promoter (SEQ ID NO: 4) and a human progranulin coding sequence with GRN intron 5 (SEQ ID NO: 27; “Jet intron 5”), a JeT promoter (SEQ ID NO: 4) and a human progranulin coding sequence with GRN intron 9 (SEQ ID NO: 12; “Jet intron 9”), a JeT promoter (SEQ ID NO: 4) and a human progranulin coding sequence with GRN intron 12 (SEQ ID NO: 29; “Jet intron 12”), an RSV promoter (SEQ ID NO: 3) and a human progranulin coding sequence with GRN intron 5 (SEQ ID NO: 27; “RSV intron 5”), an RSV promoter (SEQ ID NO: 3) and a human progranulin coding sequence with GRN intron 9 (SEQ ID NO: 12; “RSV intron 9”), an RSV promoter (SEQ ID NO: 3) and a human progranulin coding sequence with GRN intron 11 (SEQ ID NO: 29; “RSV intron 11”), or an RSV promoter (SEQ ID NO: 3) and a human progranulin coding sequence with GRN intron 12 (SEQ ID NO: 29; “RSV intron 12”).

[0100] FIG. 15A schematically illustrates an expression plasmid containing a GRN expression cassette and a normalization expression cassette used in FIG. 15B. The GRN expression cassette includes, from 5’ to 3’, a promoter, a 5’ untranslated region containing an intron, a progranulin coding sequence (“progranulin CDS”; SEQ ID NO: 10), and a polyadenylation signal (“polyA sig”). The normalization expression cassette includes, from 5’ to 3’, a CMV promoter (SEQ ID NO: 2), a luciferase coding sequence (“Sec. Luciferase”), and a polyadenylation signal (“polyA sig”).

[0101] FIG. 15B shows ELISA data of progranulin expression fold change over luciferase activity from cells transfected with the expression plasmid of FIG. 15A including a GRN expression cassette containing a 5’ untranslated region with no intron, a 5’ untranslated region with a GRN intron 11 (SEQ ID NO: 28, “GRN11”), a 5’ untranslated region with a GRN intron 12 (SEQ ID NO: 29, “GRN12”), a 5’ untranslated region with a GRN intron 9 (SEQ ID NO: 12, “GRN9”), a 5’ untranslated region with an SV40 intron (SEQ ID NO: 13, “SV40-99”), a 5’ untranslated region with an SV40 smT intron (SEQ ID NO: 30, “SVsmT”), or a 5’ untranslated region with a TPI intron 3 (SEQ ID NO: 31, “TPI3”). Expression was evaluated in HEK cells using an RSV promoter (SEQ ID NO: 3). Circles and triangles show a first replicate and a second replicate, respectively, of each assay.

[0102] FIG. 16A shows ELISA data of progranulin expression from HEK293 cells infected with 20,000 vg/cell of AAV9 virions encapsidating recombinant polynucleotides encoding progranulin. Cells were infected with scAAV9 encapsidating a JeT promoter (SEQ ID NO: 4) operably linked to GRN coding sequence (SEQ ID NO: 6; “scAAV9 JET PGRN”), scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6; “scAAV9 RSV PGRN”), scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6) and SV40 intron (SEQ ID NO: 13; “scAAV9 RSV SV40intron99”), scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence containing GRN intron 9 (SEQ ID NO: 11; “scAAV9 RSV PGRN int9”), ssAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6; “ssAAV9 RSV PGRN”), ssAAV9 encapsidating a CAG promoter (SEQ ID NO: 1) operably linked to GRN coding sequence (SEQ ID NO: 6) and WPRE sequence (SEQ ID NO: 7; “ssAAV9 CAG PGRN WPRE”), ssAAV9 encapsidating a hSyn promoter (SEQ ID NO: 5) operably linked to GRN coding sequence (SEQ ID NO: 6) and WPRE sequence (SEQ ID NO: 7; “ssAAV9 hSyn PGRN WPRE”), or ssAAV9 encapsidating a CMV promoter (SEQ ID NO: 2) operably linked to GRN coding sequence (SEQ ID NO: 6) and WPRE sequence (SEQ ID NO: 7; “ssAAV9 CMV PGRN WPRE”).

[0103] FIG. 16B shows ELISA data of progranulin expression from HEK293 cells infected with 60,000 vg/cell of AAV9 virions encapsidating recombinant polynucleotides encoding progranulin. Cells were infected with scAAV9 encapsidating a JeT promoter (SEQ ID NO: 4) operably linked to GRN coding sequence (SEQ ID NO: 6; “scAAV9 JET PGRN”), scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6; “scAAV9 RSV PGRN”), scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6) and SV40 intron (SEQ ID NO: 13; “scAAV9 RSV SV40intron99”), scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence containing GRN intron 9 (SEQ ID NO: 11; “scAAV9 RSV PGRN int9”), ssAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6; “ssAAV9 RSV PGRN”), ssAAV9 encapsidating a CAG promoter (SEQ ID NO: 1) operably linked to GRN coding sequence (SEQ ID NO: 6) and WPRE sequence (SEQ ID NO: 7; “ssAAV9 CAG PGRN WPRE”), ssAAV9 encapsidating a hSyn promoter (SEQ ID NO: 5) operably linked to GRN coding sequence (SEQ ID NO: 6) and WPRE sequence (SEQ ID NO: 7; “ssAAV9 hSyn PGRN WPRE”), or ssAAV9 encapsidating a CMV promoter (SEQ ID NO: 2) operably linked to GRN coding sequence (SEQ ID NO: 6) and WPRE sequence (SEQ ID NO: 7; “ssAAV9 CMV PGRN WPRE”).

[0104] FIG. 17A schematically illustrates an expression plasmid containing a GRN expression cassette and a normalization expression cassette separated by an insulator, which was used in FIG. 17B. The GRN expression cassette includes, from 5’ to 3’, an RSV promoter (SEQ ID NO: 3), a progranulin coding sequence (“progranulin CDS”), optionally, WPRE (SEQ ID NO: 7) or WPRE3 (SEQ ID NO: 8) (collectively shown as “WPRE(3)”), and a rabbit beta globin polyadenylation signal (“RBG Poly A”; SEQ ID NO: 42). The normalization expression cassette includes, from 5’ to 3’, a CMV promoter (SEQ ID NO: 2), a luciferase coding sequence (“Sec. Luciferase”), and a polyadenylation signal (“polyA sig”).

[0105] FIG. 17B shows ELISA data of progranulin expression fold change normalized to luciferase activity from H4 GRN KO cells transfected with the expression plasmid of FIG. 17A including a GRN expression cassette, wherein, from left to right, the progranulin CDS is SEQ ID NO: 10 and no WPRE or WPRE3 (“-”); the progranulin CDS is SEQ ID NO: 10 and the WPRE is SEQ ID NO: 7 (“WPRE”); the progranulin CDS is SEQ ID NO: 10 and the WPRE3 is SEQ ID NO: 8 (“WPRE3”); the progranulin CDS is SEQ ID NO: 14 and no WPRE or WPRE3 (“SV40in99-”); the progranulin CDS is SEQ ID NO: 14 and the WPRE is SEQ ID NO: 7 (“SV40int99 WPRE”); the progranulin CDS is SEQ ID NO: 14 and the WPRE is SEQ ID NO: 8 (“SV40int99WPRE3”); and the progranulin CDS is SEQ ID NO: 11 and WPRE is SEQ ID NO: 8 (“endo GRNint9 WPRE”).

[0106] FIG. 18A shows a bar graph of progranulin expression (in pg/mL) in neurons derived from human induced pluripotent stem cells (iPSCs) treated with AAV vectors containing either a GRN expression cassette with an RSV promoter (SEQ ID NO: 3), a progranulin coding sequence, a WPRE3 (SEQ ID NO: 8), and a rabbit beta globin polyadenylation signal (SEQ ID NO: 42) (“RSV + WPRE3”), or a GRN expression cassette with an RSV promoter (SEQ ID NO: 3), a progranulin coding sequence, a WPRE (SEQ ID NO: 7), and a rabbit beta globin polyadenylation signal (SEQ ID NO: 42) (“RSV + WPRE”). Each GRN expression cassette also included a normalization expression cassette containing a CMV promoter (SEQ ID NO: 2), a luciferase coding sequence, and a polyadenylation signal. Progranulin expression was measured on days 3 and 7 following AAV treatment.

[0107] FIG. 18B shows a bar graph of progranulin expression (in pg/mL) in mouse primary neurons treated with AAV vectors containing either a GRN expression cassette with an RSV promoter (SEQ ID NO: 3), a progranulin coding sequence, a WPRE3 (SEQ ID NO: 8), and a rabbit beta globin polyadenylation signal (SEQ ID NO: 42) (“RSV + WPRE3”), or a GRN expression cassette with an RSV promoter (SEQ ID NO: 3), a progranulin coding sequence, a WPRE (SEQ ID NO: 7), and a rabbit beta globin polyadenylation signal (SEQ ID NO: 42) (“RSV + WPRE”). Each GRN expression cassette also included a normalization expression cassette containing a CMV promoter (SEQ ID NO: 2), a luciferase coding sequence, and a polyadenylation signal. Progranulin expression was measured on days 3 and 7 following AAV treatment.

[0108] FIG. 19 provides viable cell density and percent viability of stable cell lines transfected with different payload constructs.

[0109] FIG. 20 provides titers of rAAV encapsidating a sequence encoding progranulin (vg/ml) and titers of rAAV capsid proteins (vp/ml) produced by stable cell lines transfected with different payload constructs.

[0110] FIG. 21 shows titers of viral genome (vg)/ml, viral particles (vp)/ml, and Rep proteins (Rep)/ml produced by clones derived from stable cell line pool T205- RSV Promoter.

[0111] FIG. 22 shows high titer produced after induction of single clones from the stable cell line pool T205. The graph shows the capsid titer (vp/ml) from cell lysate produced after induction of single clones (from the stable cell line pool T205) compared to the stable cell line pool T205 after induction and compared to titer produced from cells after transient transfection. The top clone shows a greater than 10-fold improvement in viral titer over transient transfection titer, indicating that similarly to select single cell clones from the T42 pool stable cell line with an eGFP payload (e.g., Clone D (eGFP)), higher titer can be achieved after induction of select single cell clones from a pool stable cell line (here, from the stable cell line pool T205) having a therapeutically relevant payload (here, a sequence coding for progranulin (PGRN)) compared to transient transfection titer. Titer was measured from AAV5-sc-payload lysate pre-purification by capsid ELISA for viral particle.

[0112] FIG. 23 shows progranulin production (PGRN pg/mL) by cells infected at the indicated multiplicity of infection of rAAV virion titers (MOI vg/cell) that were produced after induction of a single cell clone cell line (CL23) selected from the stable cell line pool T205.

[0113] FIG. 24A shows a bar plot of brain RNA levels of human progranulin in mice after injection with expression cassettes having a promoter comprising an RSV promoter (“RSV”, SEQ ID NO: 65) or a human synapsin promoter (“hSyn”).

[0114] FIG. 24B shows a bar plot of expression of human progranulin in the cerebrospinal fluid (CSF) of mice after injection with expression cassettes having an RSV promoter (“RSV”, SEQ ID NO: 65) or a human synapsin promoter (“hSyn”). [0115] FIG. 25A shows a bar plot of secreted progranulin (PGRN) levels that were secreted from H4 progranulin knockout cells infected with scAAV virions containing progranulin expression cassettes encoding progranulin under transcriptional control of a JeT promoter (“JeT”), progranulin under transcriptional control of an RSV promoter (“RSV”), progranulin under transcriptional control of an RSV promoter and having an SV40 intron in the 5’UTR (“RSV_SV40intron”), or progranulin comprising intron 9 under transcriptional control of an RSV promoter (“RSV_endo_intm9”).

[0116] FIG. 25B shows a bar plot of secreted progranulin (GRN) levels that were secreted from H4 progranulin knockout cells transfected with AAV plasmids comprising progranulin expression cassettes encoding progranulin under control of an RSV promoter (“scAAV RSV”) or progranulin under control of an RSV promoter and having an S V40 intron in the 5 ’UTR (“scAAV_RSV_SV40intron99”), or ssAAV plasmids comprising progranulin expression cassettes encoding progranulin under control of an RSV promoter (“ssAAV RSV”), progranulin under control of a CAG promoter (“ssAAV CAG”), progranulin under control of a synapsin promoter (“ssAAV hSyn”), or progranulin under control of a CMV promoter (“ssAAV_CMV”).

[0117] FIG. 26 shows a block diagram of a recombinant polynucleotide of SEQ ID NO: 65. The recombinant polynucleotide of SEQ ID NO: 65 includes, from 5’ to 3’, a 5’ inverted terminal repeat (“5’ ITR”) of SEQ ID NO: 59, a 5’ stuffer sequence (“5’ Stuffer”) of SEQ ID NO: 61, an RSV promoter (“RSV”) of SEQ ID NO: 3, a 5’ untranslated region (“5’ UTR”) of SEQ ID NO: 62 (which overlaps with the RSV promoter and comprises 33 nucleotides of the 3’ end of the RSV promoter (overlap not shown) and a Kozak sequence of SEQ ID NO: 66), a progranulin coding sequence (“GRN Coding Sequence”) of SEQ ID NO: 10 (comprising a sequence of SEQ ID NO: 57 encoding a progranulin signal peptide and a sequence of SEQ ID NO: 58), a Hindlll restriction site, a WPRE3 post-transcriptional regulatory element (“WPRE3”) of SEQ ID NO: 8, a Xball restriction site, a polyadenylation signal (“Poly A”) of SEQ ID NO: 42, a Notl restriction site, a transcriptional pause site (“Pause Site”) of SEQ ID NO: 64, a Sall restriction site, a PstI restriction site, and a 3’ inverted terminal repeat (“3’ ITR”) of SEQ ID NO: 60.

[0118] FIG. 27A shows a plot comparing viral genomes per diploid genome (vg/dg) in brain tissue extracted from mice infected with scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6) (“scAAV9 RSV”), scAAV9 encapsidating a JeT promoter (SEQ ID NO: 4) operably linked to GRN coding sequence (SEQ ID NO: 6) (“scAAV9 JET”), scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6) and SV40 intron (SEQ ID NO: 13) (“scAAV9 RSV SV40 intron 99”), scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence containing GRN intron 9 (SEQ ID NO: 11) (“scAAV9 RSV intron 9”), ssAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6) (“ssAAV9 RSV”), ssAAV9 encapsidating a CAG promoter (SEQ ID NO: 1) operably linked to GRN coding sequence (SEQ ID NO: 6) (“ssAAV9 CAG”), ssAAV9 encapsidating a hSyn promoter (SEQ ID NO: 5) operably linked to GRN coding sequence (SEQ ID NO: 6) (“ssAAV9 hSyn”), or ssAAV9 encapsidating a CMV promoter (SEQ ID NO: 2) operably linked to GRN coding sequence (SEQ ID NO: 6) (“ssAAV9 CMV”).

[0119] FIG. 27B shows a plot comparing viral genomes per diploid genome (vg/dg) in liver tissue extracted from mice infected with scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6) (“scAAV9 RSV”), scAAV9 encapsidating a JeT promoter (SEQ ID NO: 4) operably linked to GRN coding sequence (SEQ ID NO: 6) (“scAAV9 JET”), scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6) and SV40 intron (SEQ ID NO: 13) (“scAAV9 RSV SV40 intron 99”), scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence containing GRN intron 9 (SEQ ID NO: 11) (“scAAV9 RSV intron 9”), ssAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6) (“ssAAV9 RSV”), ssAAV9 encapsidating a CAG promoter (SEQ ID NO: 1) operably linked to GRN coding sequence (SEQ ID NO: 6) (“ssAAV9 CAG”), ssAAV9 encapsidating a hSyn promoter (SEQ ID NO: 5) operably linked to GRN coding sequence (SEQ ID NO: 6) (“ssAAV9 hSyn”), or ssAAV9 encapsidating a CMV promoter (SEQ ID NO: 2) operably linked to GRN coding sequence (SEQ ID NO: 6) (“ssAAV9 CMV”).

DETAILED DESCRIPTION

[0120] The present disclosure provides a recombinant polynucleotide for expressing a payload (e.g., a transgene). A recombinant polynucleotide of the present disclosure can be used to treat a disease or disorder caused by a mutation, deletion, or altered expression of a gene. For example, a recombinant polynucleotide can be used to treat a disease or disorder caused by a mutation, deletion, or altered expression of a gene by expressing a wild type copy of the gene encoded by the recombinant polynucleotide. Various sequence elements, such as promoters, introns, post- transcriptional regulatory elements, transcriptional pause sites, or polyadenylation signals can affect transcriptional levels of a payload encoded by the recombinant polynucleotide. Described herein are sequence elements and recombinant polynucleotides comprising sequence elements for expression of a payload (e.g., a progranulin payload). Variation of the sequence elements included in a recombinant polynucleotide can be used to tune expression levels, locations (e.g., cells, tissues, or secreted fluids), or both of a payload encoded by the recombinant polynucleotide.

[0121] A recombinant polynucleotide can comprise a promoter and a coding sequence under transcriptional control of the promoter. For example, the coding sequence encodes a progranulin protein and the promoter is an RSV promoter. In some embodiments, the recombinant polynucleotide includes additional sequence elements that enhance transcription of the payload. Examples of additional sequence elements that can be included in a recombinant polynucleotide are an intron, a polyadenylation signal (polyA signal) encoding a signaling addition of a polyadenylation tail (polyA tail), a post-transcriptional regulatory element, a transcriptional pause site, a neuron-restrictive silencer element (NRSE), a CCCTC-binding factor (CTCF) sequence, or any combination thereof. For example, a recombinant polynucleotide includes an RSV promoter, a coding sequence encoding progranulin, a post-transcriptional regulatory element, a polyadenylation signal, and a transcriptional pause site. A coding sequence encoding the payload can be operably linked to a promoter sequence, such that the promoter regulates expression of the payload. The payload can be a progranulin. In some embodiments, the promoter promotes different levels of payload expression in different cell types, tissue types, organs, or body regions. For example, a promoter promotes different levels of payload expression in a cerebrospinal fluid compared to in a serum of a subject administered the recombinant polynucleotide.

[0122] A neuron-restrictive silencer element (NRSE) can be positioned in the recombinant polynucleotide for payload expression such that the NRSE regulates cell specificity of payload expression. For example, an NRSE is located either upstream or downstream of the promoter. In some embodiments, the NRSE enhances expression of the payload in neuronal cells. In some embodiments, the NRSE silences expression of the payload in non-neuronal cells. A transcriptional pause site can be positioned in the recombinant polynucleotide for payload expression such that the transcriptional pause site reduces transcriptional silencing. The transcriptional pause site can reduce transcriptional silencing caused by transcriptional readthrough from an upstream promoter. In some embodiments, a transcriptional pause site is located downstream of the coding sequence. In some embodiments, the transcriptional pause site enhances expression of the payload. A CCCTC-binding factor (CTCF) sequence can be positioned in the recombinant polynucleotide for payload expression such that the CTCF modifies accessibility of the recombinant polynucleotide to transcription machinery to enhance expression of the payload. An intron can be positioned in a recombinant polynucleotide for payload expression such that the intron regulates expression of the payload. For example, an intron is located in the 5’UTR of the sequence encoding the payload, such as an SV40 intron in the 5’UTR. In some embodiments, the coding sequence of the payload comprises a small intron and no other introns. For example, the coding sequence of progranulin comprises no introns except for intron 9 (e.g., a sequence of progranulin with no introns except for intron is SEQ ID NO: 11). A sequence element can be included in the recombinant polynucleotide such that the sequence element regulates expression of the payload. For example, a sequence element comprises a post-transcriptional regulatory element, such as a woodchuck hepatitis virus post- transcriptional regulatory element (WPRE), that is positioned downstream of the sequence encoding the payload and proximal to the sequence encoding a polyadenylation signal. A polyadenylation (poly A) signal sequence can be included in a recombinant polynucleotide for payload expression such that the polyA signal sequence regulates expression of the payload. For example, the polyA sequence is a rabbit beta globulin polyA sequence. These different parts of the recombinant polynucleotide can be combined to result in the desired level of expression of the payload in cell type, tissue type, organ, or body region of interest. Differential payload expression across cell types, tissue types, organs, or body regions can facilitate treatment of a disease or disorder by expressing the payload in a cell, tissue, organ, or body region associated with the disease or disorder. For example, the payload is expressed in a fluid (e.g., a cerebrospinal fluid) secreted by a cell or tissue. In some embodiments, the different parts of the recombinant polynucleotide are combined to produce robust progranulin expression in multiple cell lines (e.g., glioblastoma cell lines, neuronal cell lines, microglia cell lines, and/or HEPG2 cell lines).

[0123] In some embodiments, a recombinant polynucleotide of the present disclosure comprises a promoter operably linked to a sequence encoding the payload. In some embodiments, the recombinant polynucleotide is for expressing progranulin (PGRN). In some embodiments, the recombinant polynucleotide comprises a GRN sequence for expression of progranulin. In some embodiments, the recombinant polynucleotide comprises a promoter operably linked to a GRN sequence. In some embodiments, the promoter is a CAG promoter, a CMV promoter, an RSV promoter, a JeT promoter, or a Synapsin promoter. For example, the promoter is an RSV promoter. The promoter can be selected to promote high levels of progranulin expression in cerebrospinal fluid. The promoter can be selected to promote high levels of progranulin expression in multiple cell lines (e.g., glioblastoma cell lines, neuronal cell lines, and/or microglia cell lines). The promotor can be selected for a short nucleotide sequence while promoting high levels of progranulin expression in cerebrospinal fluid and/or multiple cell lines. For example, the promoter has a nucleotide sequence of less than 300 nucleotide bases.

[0124] In some embodiments, the recombinant polynucleotide comprises a neuron-restrictive silencer element (NRSE). A NRSE can be located either upstream or downstream of the promoter. In some embodiments, the NRSE enhances expression of the payload in neuronal cells. In some embodiments, the NRSE silences expression of the payload in non-neuronal cells. In some embodiments, the recombinant polynucleotide comprises a CCCTC-binding factor (CTCF) sequence. A CTCF sequence can modify accessibility of the recombinant polynucleotide to transcription machinery to enhance expression of the payload. In some embodiments, the recombinant polynucleotide is engineered to comprise an intron for increased expression of the payload (e.g., progranulin), such as intron 9 of GRN or an intron from SV40. For example, an intron of the recombinant polynucleotide is engineered to enhance expression of the GRN sequence. The GRN sequence can comprise no introns. The GRN sequence can comprise only exons. In some embodiments, the GRN sequence comprises a small intron. A small intron can be an intron comprising no more than 200 nucleotides. In some embodiments, the small intron comprises from 30 to 200 nucleotides in length. In some embodiments, the small intron comprises from 50 to 200 nucleotides in length. In some embodiments, the small intron comprises from 50 to 150 nucleotides in length. In some embodiments, the small intron comprises from 70 to 120 nucleotides in length. In some embodiments, the small intron comprises about 120 nucleotides in length. The GRN sequence can comprise intron 9 of the GRN sequence and no other introns. In some embodiments, the recombinant polynucleotide comprises an SV40 intron. The SV40 intron is positioned in the 5’UTR, for example, downstream of the promoter sequence and upstream of the translational start site of the sequence encoding the payload (e.g., progranulin). In some embodiments, the recombinant polynucleotide is engineered for increased expression of the payload (e.g., progranulin). For example, the individual sequence elements of the recombinant polynucleotide can be engineered to enhance expression of the GRN sequence.

[0125] In some embodiments, the recombinant polynucleotide comprises an element that increases expression of the payload (e.g., progranulin), such as a post-transcriptional regulatory element. In some embodiments, the post-transcriptional regulatory element is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) or a variant thereof (e.g., a shortened WPRE, WPRE3). In some embodiments, the recombinant polynucleotide is engineered to comprise a polyadenylation (poly A) signal for increased expression of the payload (e.g., progranulin). For example, the recombinant polynucleotide is engineered to comprise a rabbit beta globulin polyA sequence. In some embodiments the recombinant polynucleotide is engineered to comprise an element that reduces transcriptional silencing, such as a transcriptional pause site. In some embodiments, a transcriptional pause site may comprise a polyA signal (e.g., a second polyA signal). The transcriptional pause site may reduce transcriptional silencing caused by transcriptional readthrough from an upstream promoter. [0126] In some embodiments, the recombinant polynucleotide is in a vector. The vector can be a plasmid. The vector can be a viral vector. The viral vector can be an adeno-associated virus (AAV) vector. An AAV can be single stranded AAV (ssAAV) or self-complementary AAV (scAAV). The AAV can be an AAV9 encapsidating the recombinant polynucleotide, wherein the recombinant polynucleotide comprises a promoter operably linked to a GRN sequence for the expression of progranulin. In some embodiments, the GRN sequence comprises no introns. In some embodiments, the GRN sequence comprises only introns. In some embodiments, the recombinant polynucleotide comprises an intron. The intron can a GRN intron, such as intron 9. Intron 9 can be located in the GRN sequence. The intron can be an SV40 intron. The SV40 intron can be located downstream of the promoter sequence and upstream of the translational start site of the GRN sequence. In some embodiments, the recombinant polynucleotide further comprises sequence element, such as a WPRE or variant thereof. In some embodiments, the recombinant polynucleotide further comprises a polyadenylation signal sequence, such as a rabbit beta globulin polyA sequence. The recombinant polynucleotide can be delivered via viral vector to a subject in need thereof. In some embodiments, the subject in need thereof has a disease or disorder, such as frontotemporal dementia (FTD), Amyotrophic Lateral Sclerosis (ALS), Alzheimer’s Disease, Parkinson’s Disease, or dementia.

[0127] The recombinant polynucleotide can be used to treat a disease or disorder. In some embodiments, the recombinant polynucleotide can be delivered via viral vector to treat a disease or disorder to a subject in need thereof. For example, the recombinant polynucleotide comprises a promoter operably linked to a GRN sequence for the expression of progranulin and is used to treat frontotemporal dementia (FTD). The progranulin can be expressed in a cerebrospinal fluid of a subject to treat the frontotemporal dementia in the subject. The cerebrospinal fluid is secreted by a cell of tissue expressing the progranulin. In some embodiments, the GRN sequence comprises no introns. In some embodiments, the GRN sequence comprises all native GRN introns. In some embodiments, the GRN sequence comprises only introns. In some embodiments, the GRN sequence comprises one or more introns (e.g., one or more native GRN introns). In some embodiments, the recombinant polynucleotide comprises an intron. The intron can a GRN intron, such as intron 9. Intron 9 can be located in the GRN sequence. The intron can be an SV40 intron. The SV40 intron can be located downstream of the promoter sequence and upstream of the translational start site of the GRN sequence. In some embodiments, the recombinant polynucleotide comprises a NRSE. In some embodiments, the recombinant polynucleotide comprises a CTCF sequence. In some embodiments, the recombinant polynucleotide further comprises sequence element, such as a WPRE or variant thereof. In some embodiments, the recombinant polynucleotide further comprises a polyadenylation signal sequence, such as a rabbit beta globulin polyA sequence.

Recombinant Polynucleotides

[0128] A recombinant polynucleotide of the present disclosure can include a promoter and a coding sequence (e.g., a sequence encoding a payload). The recombinant polynucleotide can comprise a polynucleotide cassette coding for expression of a coding sequence, also referred to as an “expression cassette”. An expression cassette can comprise a coding sequence (e.g., a progranulin coding sequence) and one or more regulatory sequences (e.g., a promoter, a 5’ UTR, a post-transcriptional regulatory element, a polyadenylation signal, or combinations thereof) operably linked to the coding sequence to regulate transcription of the coding sequence. The promoter can be operably linked to the coding sequence. For example, the coding sequence is under transcriptional control of the promoter. The payload can be progranulin, and therefore the sequence encoding the payload can be a sequence encoding progranulin (e.g., a GRN sequence, such as SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 26 encoding a progranulin protein of SEQ ID NO: 63). In some embodiments, the recombinant polynucleotide further comprises additional elements (e.g., a post-transcriptional regulatory element, a polyadenylation signal, a transcriptional pause site, a neuron-restrictive silencer element, a CCCTC-binding factor sequence, a 5’ untranslated region, a 3’ untranslated region, a 5’ stuffer sequence, or combinations thereof). The additional elements can enhance or increase expression of the payload. For example, the additional elements can enhance or increase transcription or translation of the GRN sequence.

[0129] In some embodiments, it is advantageous to include small components (e.g., components with relatively short polynucleotide sequences) in a recombinant polynucleotide. Since delivery vectors (e.g., viral vectors or plasmids) can have a limited polynucleotide capacity, inclusion of small components rather than larger components (e.g., components with longer polynucleotide sequences) in a recombinant polynucleotide for delivery by the vector can leave room in the vector for additional elements. For example, inclusion of small components in a recombinant polynucleotide delivered by a vector can leave room in the vector for an additional coding sequence, an additional regulatory sequence, or an additional expression cassette. An example of a small promoter is an RSV promoter (e.g., SEQ ID NO: 3). An example of a small post- transcriptional regulatory element is a WPRE3 (e.g., SEQ ID NO: 8).

Promoter

[0130] The promoter of the recombinant polynucleotide can be operably linked to the sequence encoding the payload. In some embodiments, the promoter comprises an enhancer, a core promoter, or combinations thereof. The promoter can recruit transcription factors, polymerases (e.g., RNA polymerase II or RNA polymerase III), or other transcriptional machinery to promote transcription of the payload. For example, the promoter of the recombinant polynucleotide promotes transcription of the GRN sequence. In some embodiments, an enhancer recruits additional factors (e.g., transcription factors) that enhance transcription of the payload. In some embodiments, a promoter is a constitutive promoter (e.g., a promoter that promotes transcription in a wide variety of tissues and cell types). In some embodiments, a promoter with a relatively short sequence is advantageous to leave room for other sequences (e.g., coding sequence or additional regulatory elements) within a delivery vector (e.g., an AAV). For example, a promoter has a sequence that is not more than about 600 nucleotides in length, not more than 500 nucleotides in length, not more than 300 nucleotides in length, or not more than 275 nucleotides in length. In some embodiments, a promoter is selected or engineered for reduced transcriptional silencing in a cell or tissue of interest (e.g., reduced transcriptional silencing in neurons). In some embodiments, a promoter is selected or engineered for high payload expression in a cell or tissue of interest (e.g., high payload expression in neurons or high payload expression in a cerebrospinal fluid). In some embodiments, a promoter is selected or engineered for low payload expression in other cells or tissues (e.g., low payload expression in kidneys or low payload expression in a serum).

[0131] In some embodiments, a promoter is a specific promoter (e.g., a neuron-specific promoter). In some embodiments, a promoter is a constitutive promoter. A promoter can be a CAG promoter. A promoter can be a CMV promoter. A promoter can be an RSV promoter. A promoter can be a JeT promoter. A promoter can be a Synapsin promoter. In some embodiments, the Synapsin promoter can be a human Synapsin promoter (hSynapsin). In some embodiments, a promoter promotes different levels of payload expression in different cell types, tissue types, organs, or body regions. For example, the promoter is a neuron-specific promoter (e.g., a synapsin promoter). In some embodiments, the promoter promotes expression of the payload in cerebrospinal fluid. In some embodiments, the promoter promotes expression of the payload in multiple cell lines (e.g., glioblastoma cell lines, neuronal cell lines, and/or microglia cell lines). Exemplary promoter sequences that can be operably linked to a sequence of a payload are provided in TABLE 1.

TABLE 1. Exemplary Promoter Sequences

[0132] In preferred embodiments, the promoter is an RSV promoter. An engineered polynucleotide of the present disclosure can comprise a coding sequence (e.g., a sequence encoding progranulin) operably linked to an RSV promoter. An engineered polynucleotide can comprise a coding sequence (e.g., a sequence encoding progranulin) under transcriptional control of an RSV promoter. An engineered polynucleotide comprising an RSV promoter can further comprise one or more additional elements (e.g., a post-transcriptional regulatory element, a polyadenylation signal, a transcriptional pause site, a neuron-restrictive silencer element, a CCCTC-binding factor sequence, a 5’ untranslated region, a 3’ untranslated region, a 5’ stuffer sequence, or combinations thereof). An RSV promoter can function as a constitutive promoter. In some embodiments, the RSV promoter has less transcriptional silencing than other constitutive promoters. In some embodiments, the RSV promoter promotes robust transcription of the coding sequence in neurons. In some embodiments, the RSV promoter enhances payload expression in cerebrospinal fluid. The RSV promoter may be selected due to its short nucleotide sequence compared to other promoters. The RSV promoter can comprise at least 80% sequence identity to SEQ ID NO: 3. The RSV promoter can comprise at least 85% sequence identity to SEQ ID NO: 3. The RSV promoter can comprise at least 90% sequence identity to SEQ ID NO: 3. The RSV promoter can comprise at least 95% sequence identity to SEQ ID NO: 3. The RSV promoter can comprise at least 96% sequence identity to SEQ ID NO: 3. The RSV promoter can comprise at least 97% sequence identity to SEQ ID NO: 3. The RSV promoter can comprise at least 98% sequence identity to SEQ ID NO: 3. The RSV promoter can comprise at least 99% sequence identity to SEQ ID NO: 3. The RSV promoter can comprise 100% sequence identity to SEQ ID NO: 3. The recombinant polynucleotide can comprise a RSV promoter having a sequence of SEQ ID NO: 3. In some embodiments, the RSV promoter is upstream of the sequence encoding the payload in the recombinant polynucleotide. In some embodiments, the RSV promoter is upstream of the sequence encoding progranulin in the recombinant polynucleotide.

[0133] In some embodiments, the recombinant polynucleotide comprises a CAG promoter. The CAG promoter can comprise at least 80% sequence identity to SEQ ID NO: 1. The CAG promoter can comprise at least 85% sequence identity to SEQ ID NO: 1. The CAG promoter can comprise at least 90% sequence identity to SEQ ID NO: 1. The CAG promoter can comprise at least 95% sequence identity to SEQ ID NO: 1. The CAG promoter can comprise at least 96% sequence identity to SEQ ID NO: 1. The CAG promoter can comprise at least 97% sequence identity to SEQ ID NO: 1. The CAG promoter can comprise at least 98% sequence identity to SEQ ID NO: 1. The CAG promoter can comprise at least 99% sequence identity to SEQ ID NO: 1. The CAG promoter can comprise 100% sequence identity to SEQ ID NO: 1. The recombinant polynucleotide can comprise a CAG promoter having a sequence of SEQ ID NO: 1. In some embodiments, the CAG promoter is upstream of the sequence encoding the payload in the recombinant polynucleotide. In some embodiments, the CAG promoter is upstream of the sequence encoding progranulin in the recombinant polynucleotide.

[0134] In some embodiments, the recombinant polynucleotide comprises a CMV promoter. The CMV promoter can comprise at least 80% sequence identity to SEQ ID NO: 2. The CMV promoter can comprise at least 85% sequence identity to SEQ ID NO: 2. The CMV promoter can comprise at least 90% sequence identity to SEQ ID NO: 2. The CMV promoter can comprise at least 95% sequence identity to SEQ ID NO: 2. The CMV promoter can comprise at least 96% sequence identity to SEQ ID NO: 2. The CMV promoter can comprise at least 97% sequence identity to SEQ ID NO: 2. The CMV promoter can comprise at least 98% sequence identity to SEQ ID NO: 2. The CMV promoter can comprise at least 99% sequence identity to SEQ ID NO: 2. The CMV promoter can comprise 100% sequence identity to SEQ ID NO: 2. The recombinant polynucleotide can comprise a CMV promoter having a sequence of SEQ ID NO: 2. In some embodiments, the CMV promoter is upstream of the sequence encoding the payload in the recombinant polynucleotide. In some embodiments, the CMV promoter is upstream of the sequence encoding progranulin in the recombinant polynucleotide.

[0135] In some embodiments, the recombinant polynucleotide comprises a JeT promoter. The JeT promoter can comprise at least 80% sequence identity to SEQ ID NO: 4. The JeT promoter can comprise at least 85% sequence identity to SEQ ID NO: 4. The JeT promoter can comprise at least 90% sequence identity to SEQ ID NO: 4. The JeT promoter can comprise at least 95% sequence identity to SEQ ID NO: 4. The JeT promoter can comprise at least 96% sequence identity to SEQ ID NO: 4. The JeT promoter can comprise at least 97% sequence identity to SEQ ID NO: 4. The JeT promoter can comprise at least 98% sequence identity to SEQ ID NO: 4. The JeT promoter can comprise at least 99% sequence identity to SEQ ID NO: 4. The JeT promoter can comprise 100% sequence identity to SEQ ID NO: 4. The recombinant polynucleotide can comprise a JeT promoter having a sequence of SEQ ID NO: 4. In some embodiments, the JeT promoter is upstream of the sequence encoding the payload in the recombinant polynucleotide. In some embodiments, the JeT promoter is upstream of the sequence encoding progranulin in the recombinant polynucleotide.

[0136] In some embodiments, the recombinant polynucleotide comprises a Synapsin promoter. In some embodiments, the recombinant polynucleotide comprises a hSypnasin promoter. The hSypnasin promoter can comprise at least 80% sequence identity to SEQ ID NO: 5. The hSypnasin promoter can comprise at least 85% sequence identity to SEQ ID NO: 5. The hSypnasin promoter can comprise at least 90% sequence identity to SEQ ID NO: 5. The hSypnasin promoter can comprise at least 95% sequence identity to SEQ ID NO: 5. The hSypnasin promoter can comprise at least 96% sequence identity to SEQ ID NO: 5. The hSypnasin promoter can comprise at least 97% sequence identity to SEQ ID NO: 5. The hSypnasin promoter can comprise at least 98% sequence identity to SEQ ID NO: 5. The hSypnasin promoter can comprise at least 99% sequence identity to SEQ ID NO: 5. The hSypnasin promoter can comprise 100% sequence identity to SEQ ID NO: 5. The recombinant polynucleotide can comprise a hSypnasin promoter having a sequence of SEQ ID NO: 5. In some embodiments, the hSypnasin promoter is upstream of the sequence encoding the payload in the recombinant polynucleotide. In some embodiments, the hSypnasin promoter is upstream of the sequence encoding progranulin in the recombinant polynucleotide.

[0137] In some embodiments, the recombinant polynucleotide comprises a minimal CMV (CMVmin) promoter. The CMVmin promoter can comprise at least 80% sequence identity to SEQ ID NO: 21. The CMVmin promoter can comprise at least 85% sequence identity to SEQ ID NO: 21. The CMVmin promoter can comprise at least 90% sequence identity to SEQ ID NO: 21. The CMVmin promoter can comprise at least 95% sequence identity to SEQ ID NO: 21. The CMVmin promoter can comprise at least 96% sequence identity to SEQ ID NO: 21. The

CMVmin promoter can comprise at least 97% sequence identity to SEQ ID NO: 21. The

CMVmin promoter can comprise at least 98% sequence identity to SEQ ID NO: 21. The

CMVmin promoter can comprise at least 99% sequence identity to SEQ ID NO: 21. The

CMVmin promoter can comprise 100% sequence identity to SEQ ID NO: 21. The recombinant polynucleotide can comprise a CMVmin promoter having a sequence of SEQ ID NO: 21. In some embodiments, the CMVmin promoter is upstream of the sequence encoding the payload in the recombinant polynucleotide. In some embodiments, the CMVmin promoter is upstream of the sequence encoding progranulin in the recombinant polynucleotide.

[0138] In some embodiments, the recombinant polynucleotide comprises a EF1α promoter. The EF1α promoter can comprise at least 80% sequence identity to SEQ ID NO: 22. The EF1α promoter can comprise at least 85% sequence identity to SEQ ID NO: 22. The EF1α promoter can comprise at least 90% sequence identity to SEQ ID NO: 22. The EF1α promoter can comprise at least 95% sequence identity to SEQ ID NO: 22. The EF1α promoter can comprise at least 96% sequence identity to SEQ ID NO: 22. The EF1α promoter can comprise at least 97% sequence identity to SEQ ID NO: 22. The EF1α promoter can comprise at least 98% sequence identity to SEQ ID NO: 22. The EF1α promoter can comprise at least 99% sequence identity to SEQ ID NO: 22. The EF1α promoter can comprise 100% sequence identity to SEQ ID NO: 22. The recombinant polynucleotide can comprise a EF1α promoter having a sequence of SEQ ID NO: 22. In some embodiments, the EF1α promoter is upstream of the sequence encoding the payload in the recombinant polynucleotide. In some embodiments, the EF1α promoter is upstream of the sequence encoding progranulin in the recombinant polynucleotide.

[0139] In some embodiments, the recombinant polynucleotide comprises a putative MECP2 promoter or a portion of a putative MECP2 promoter (MECP2 promoter). For example, the MECP2 promoter can comprise 212 base pairs of a putative MECP2 promoter. The MECP2 promoter can comprise at least 80% sequence identity to SEQ ID NO: 23. The MECP2 promoter can comprise at least 85% sequence identity to SEQ ID NO: 23. The MECP2 promoter can comprise at least 90% sequence identity to SEQ ID NO: 23. The MECP2 promoter can comprise at least 95% sequence identity to SEQ ID NO: 23. The MECP2 promoter can comprise at least 96% sequence identity to SEQ ID NO: 23. The MECP2 promoter can comprise at least 97% sequence identity to SEQ ID NO: 23. The MECP2 promoter can comprise at least 98% sequence identity to SEQ ID NO: 23. The MECP2 promoter can comprise at least 99% sequence identity to SEQ ID NO: 23. The MECP2 promoter can comprise 100% sequence identity to SEQ ID NO: 23. The recombinant polynucleotide can comprise a MECP2 promoter having a sequence of SEQ ID NO: 23. In some embodiments, the MECP2 promoter is upstream of the sequence encoding the payload in the recombinant polynucleotide. In some embodiments, the MECP2 promoter is upstream of the sequence encoding progranulin in the recombinant polynucleotide. [0140] In some embodiments, the recombinant polynucleotide comprises a B-actin promoter, such as a chicken B-actin promoter. The B-actin promoter can comprise at least 80% sequence identity to SEQ ID NO: 24. The B-actin promoter can comprise at least 85% sequence identity to SEQ ID NO: 24. The B-actin promoter can comprise at least 90% sequence identity to SEQ ID NO: 24. The B-actin promoter can comprise at least 95% sequence identity to SEQ ID NO: 24. The B-actin promoter can comprise at least 96% sequence identity to SEQ ID NO: 24. The B- actin promoter can comprise at least 97% sequence identity to SEQ ID NO: 24. The B-actin promoter can comprise at least 98% sequence identity to SEQ ID NO: 24. The B-actin promoter can comprise at least 99% sequence identity to SEQ ID NO: 24. The B-actin promoter can comprise 100% sequence identity to SEQ ID NO: 24. The recombinant polynucleotide can comprise a B-actin promoter having a sequence of SEQ ID NO: 24. In some embodiments, the B- actin promoter is upstream of the sequence encoding the payload in the recombinant polynucleotide. In some embodiments, the B-actin promoter is upstream of the sequence encoding progranulin in the recombinant polynucleotide.

[0141] In some embodiments, the recombinant polynucleotide comprises a MND promoter. The MND promoter can comprise at least 80% sequence identity to SEQ ID NO: 25. The MND promoter can comprise at least 85% sequence identity to SEQ ID NO: 25. The MND promoter can comprise at least 90% sequence identity to SEQ ID NO: 25. The MND promoter can comprise at least 95% sequence identity to SEQ ID NO: 25. The MND promoter can comprise at least 96% sequence identity to SEQ ID NO: 25. The MND promoter can comprise at least 97% sequence identity to SEQ ID NO: 25. The MND promoter can comprise at least 98% sequence identity to SEQ ID NO: 25. The MND promoter can comprise at least 99% sequence identity to SEQ ID NO: 25. The MND promoter can comprise 100% sequence identity to SEQ ID NO: 25. The recombinant polynucleotide can comprise a MND promoter having a sequence of SEQ ID NO: 25. In some embodiments, the MND promoter is upstream of the sequence encoding the payload in the recombinant polynucleotide. In some embodiments, the MND promoter is upstream of the sequence encoding progranulin in the recombinant polynucleotide.

[0142] In some embodiments, the recombinant polynucleotide comprises a ybTATA promoter. The ybTATA promoter can comprise at least 80% sequence identity to SEQ ID NO: 54. The ybTATA promoter can comprise at least 85% sequence identity to SEQ ID NO: 54. The ybTATA promoter can comprise at least 90% sequence identity to SEQ ID NO: 54. The ybTATA promoter can comprise at least 95% sequence identity to SEQ ID NO: 54. The ybTATA promoter can comprise at least 96% sequence identity to SEQ ID NO: 54. The ybTATA promoter can comprise at least 97% sequence identity to SEQ ID NO: 54. The ybTATA promoter can comprise at least 98% sequence identity to SEQ ID NO: 54. The ybTATA promoter can comprise at least 99% sequence identity to SEQ ID NO: 54. The ybTATA promoter can comprise 100% sequence identity to SEQ ID NO: 54. The recombinant polynucleotide can comprise a ybTATA promoter having a sequence of SEQ ID NO: 54. In some embodiments, the ybTATA promoter is upstream of the sequence encoding the payload in the recombinant polynucleotide. In some embodiments, the ybTATA promoter is upstream of the sequence encoding progranulin in the recombinant polynucleotide.

[0143] In some embodiments, the promoter can be engineered for increased expression of the payload under transcriptional control of the promoter. Sequence elements within the promoter (e.g., transcription factor binding sequences, transcription initiation sequences, intron sequences, or combinations thereof) can be engineered for enhanced payload expression.

Payload. Sequence

[0144] The sequence encoding the payload of the recombinant polynucleotide can be operably linked to the promoter as described herein. A recombinant polynucleotide of the present disclosure can be used to express a peptide or protein encoded by the payload sequence. The payload sequence can encode a peptide associated with a disease. For example, the payload sequence encodes a functional copy of a peptide to treat a disease associated with a mutation in or decreased expression of the peptide. Expression of the peptide associated with the disease can treat the disease. In some embodiments, the payload sequence encodes a peptide fragment.

[0145] The payload of the recombinant polynucleotide can be progranulin. Therefore, the payload expressed from the recombinant polynucleotide can be progranulin. In some embodiments, the progranulin is mouse progranulin. In some embodiments, the progranulin is human progranulin. In some embodiments, the payload sequence is a GRN sequence. Exemplary payload sequences are shown in TABLE 2.

TABLE 2. Exemplary Payload Sequences

[0146] In some embodiments, the recombinant polynucleotide comprises a sequence encoding a payload (also referred to as a “coding sequence”), wherein the payload is progranulin (e.g., GRN sequence). In some embodiments, the recombinant polynucleotide comprises a sequence encoding a payload, wherein the payload is human progranulin (e.g., hGRN sequence). The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 80% sequence identity to SEQ ID NO: 6. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 85% sequence identity to SEQ ID NO: 6. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 90% sequence identity to SEQ ID NO: 6. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 95% sequence identity to SEQ ID NO: 6. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 96% sequence identity to SEQ ID NO: 6. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 97% sequence identity to SEQ ID NO: 6. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 98% sequence identity to SEQ ID NO: 6. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 99% sequence identity to SEQ ID NO: 6. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise 100% sequence identity to SEQ ID NO: 6. The recombinant polynucleotide can comprise a sequence encoding human progranulin (e.g., hGRN sequence) having a sequence of SEQ ID NO: 6. The recombinant polynucleotide can comprise a sequence encoding human progranulin (e.g., hGRN sequence) that is codon optimized (e.g., a codon optimized variation of SEQ ID NO: 6).

[0147] The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 80% sequence identity to SEQ ID NO: 10. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 85% sequence identity to SEQ ID NO: 10. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 90% sequence identity to SEQ ID NO: 10. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 95% sequence identity to SEQ ID NO: 10. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 96% sequence identity to SEQ ID NO:

10. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 97% sequence identity to SEQ ID NO: 10. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 98% sequence identity to SEQ ID NO: 10. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 99% sequence identity to SEQ ID NO: 10. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise 100% sequence identity to SEQ ID NO: 10. The recombinant polynucleotide can comprise a sequence encoding human progranulin (e.g., hGRN sequence) having a sequence of SEQ ID NO: 10. The recombinant polynucleotide can comprise a sequence encoding human progranulin (e.g., hGRN sequence) that is codon optimized (e.g., a codon optimized variation of SEQ ID NO: 10).

[0148] The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 80% sequence identity to SEQ ID NO: 11. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 85% sequence identity to SEQ ID NO: 11. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 90% sequence identity to SEQ ID NO: 11. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 95% sequence identity to SEQ ID NO: 11. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 96% sequence identity to SEQ ID NO:

11. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 97% sequence identity to SEQ ID NO: 11. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 98% sequence identity to SEQ ID NO: 11. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 99% sequence identity to SEQ ID NO: 11. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise 100% sequence identity to SEQ ID NO: 11. The recombinant polynucleotide can comprise a sequence encoding human progranulin (e.g., hGRN sequence) having a sequence of SEQ ID NO: 11. The recombinant polynucleotide can comprise a sequence encoding human progranulin (e.g., hGRN sequence) that is codon optimized (e.g., a codon optimized variation of SEQ ID NO: 11).

[0149] The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 80% sequence identity to SEQ ID NO: 26. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 85% sequence identity to SEQ ID NO: 26. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 90% sequence identity to SEQ ID NO: 26. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 95% sequence identity to SEQ ID NO: 26. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 96% sequence identity to SEQ ID NO: 26. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 97% sequence identity to SEQ ID NO: 26. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 98% sequence identity to SEQ ID NO: 26. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise at least 99% sequence identity to SEQ ID NO: 26. The sequence encoding human progranulin (e.g., hGRN sequence) can comprise 100% sequence identity to SEQ ID NO: 26. The recombinant polynucleotide can comprise a sequence encoding human progranulin (e.g., hGRN sequence) having a sequence of SEQ ID NO: 26. The recombinant polynucleotide can comprise a sequence encoding human progranulin (e.g., hGRN sequence) that is codon optimized (e.g., SEQ ID NO: 26).

[0150] In a preferred embodiment, the sequence encoding human progranulin (e.g., hGRN sequence) can comprise a sequence encoding a signal peptide. For example, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 26 encoding human progranulin comprise a sequence encoding a signal peptide. The signal peptide can be part of the progranulin protein (e.g., SEQ ID NO: 63) encoded by the coding sequence. The signal peptide can be encoded by a sequence having at least 80% sequence identity to SEQ ID NO: 57. The signal peptide can be encoded by a sequence having at least 85% sequence identity to SEQ ID NO: 57. The signal peptide can be encoded by a sequence having at least 90% sequence identity to SEQ ID NO: 57. The signal peptide can be encoded by a sequence having at least 95% sequence identity to SEQ ID NO: 57. The signal peptide can be encoded by a sequence having at least 96% sequence identity to SEQ ID NO: 57. The signal peptide can be encoded by a sequence having at least 97% sequence identity to SEQ ID NO: 57. The signal peptide can be encoded by a sequence having at least 98% sequence identity to SEQ ID NO: 57. The signal peptide can be encoded by a sequence having at least 99% sequence identity to SEQ ID NO: 57. The signal peptide can be encoded by a sequence having 100% sequence identity to SEQ ID NO: 57. The signal peptide can be encoded by a sequence of SEQ ID NO: 57. The sequence encoding the signal peptide can be positioned upstream of a progranulin coding sequence (e.g., SEQ ID NO: 58).

[0151] The sequence encoding human progranulin (e.g., hGRN sequence) without a sequence encoding a signal peptide can comprise at least 80% sequence identity to SEQ ID NO: 58. The sequence encoding human progranulin (e.g., hGRN sequence) without a sequence encoding a signal peptide can comprise at least 85% sequence identity to SEQ ID NO: 58. The sequence encoding human progranulin (e.g., hGRN sequence) without a sequence encoding a signal peptide can comprise at least 90% sequence identity to SEQ ID NO: 58. The sequence encoding human progranulin (e.g., hGRN sequence) without a sequence encoding a signal peptide can comprise at least 95% sequence identity to SEQ ID NO: 58. The sequence encoding human progranulin (e.g., hGRN sequence) without a sequence encoding a signal peptide can comprise at least 96% sequence identity to SEQ ID NO: 58. The sequence encoding human progranulin (e.g., hGRN sequence) without a sequence encoding a signal peptide can comprise at least 97% sequence identity to SEQ ID NO: 58. The sequence encoding human progranulin (e.g., hGRN sequence) without a sequence encoding a signal peptide can comprise at least 98% sequence identity to SEQ ID NO: 58. The sequence encoding human progranulin (e.g., hGRN sequence) without a sequence encoding a signal peptide can comprise at least 99% sequence identity to SEQ ID NO: 58. The sequence encoding human progranulin (e.g., hGRN sequence) without a sequence encoding a signal peptide can comprise 100% sequence identity to SEQ ID NO: 58. The recombinant polynucleotide can comprise a sequence encoding human progranulin (e.g., hGRN sequence) without a sequence encoding a signal peptide having a sequence of SEQ ID NO: 58.

[0152] In some embodiments, a coding sequence (e.g., a sequence encoding human progranulin) of a recombinant polynucleotide of the present disclosure encodes progranulin protein (e.g., a human progranulin). The coding sequence can encode a protein comprising at least 80% sequence identity to SEQ ID NO: 63 (MWTLVSWVALTAGLVAGTRCPDGQFCPVACCLDPGGASYSCCRPLLDKWPTTLSRHL GGPCQVDAHCSAGHSCIFTVSGTSSCCPFPEAVACGDGHHCCPRGFHCSADGRSCFQRS GNNSVGAIQCPDSQFECPDFSTCCVMVDGSWGCCPMPQASCCEDRVHCCPHGAFCDLV HTRCITPTGTHPLAKKLPAQRTNRAVALSSSVMCPDARSRCPDGSTCCELPSGKYGCCP MPNATCCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCP DGYTCCRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWMEKA PAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQHCCPQG YTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGCDQHTSCPVGQTCCPSLGGSWA CCQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEWSAQPATFLARSPHVGVKDVECG EGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAP RWDAPLRDPALRQLL). The coding sequence can encode a protein comprising at least 85% sequence identity to SEQ ID NO: 63. The coding sequence can encode a protein comprising at least 90% sequence identity to SEQ ID NO: 63. The coding sequence can encode a protein comprising at least 95% sequence identity to SEQ ID NO: 63. The coding sequence can encode a protein comprising at least 96% sequence identity to SEQ ID NO: 63. The coding sequence can encode a protein comprising at least 97% sequence identity to SEQ ID NO: 63. The coding sequence can encode a protein comprising at least 98% sequence identity to SEQ ID NO: 63. The coding sequence can encode a protein comprising at least 99% sequence identity to SEQ ID NO: 63. The coding sequence can encode a protein comprising 100% sequence identity to SEQ ID NO: 63. The coding sequence can encode a protein comprising a sequence of SEQ ID NO: 63.

[0153] In some embodiments, the sequence encoding human progranulin (e.g., h GRN sequence) is downstream of the promoter in the recombinant polynucleotide. In some embodiments, the sequence encoding human progranulin (e.g., hGRN sequence) is downstream of a CAG promoter in the recombinant polynucleotide. In some embodiments, the sequence encoding human progranulin (e.g., hGRN sequence) is downstream of a CMV promoter in the recombinant polynucleotide. In a preferred embodiment, the sequence encoding human progranulin (e.g., hGRN sequence) is downstream of an RSV promoter in the recombinant polynucleotide. In some embodiments, the sequence encoding human progranulin (e.g., hGRN sequence) is downstream of a JeT promoter in the recombinant polynucleotide. In some embodiments, the sequence encoding human progranulin (e.g., hGRN sequence) is downstream of a hSynapsin promoter in the recombinant polynucleotide. In some embodiments, the sequence encoding human progranulin (e.g., hGRN sequence) is downstream of a minimal CMV promoter in the recombinant polynucleotide. In some embodiments, the sequence encoding human progranulin (e.g., hGRN sequence) is downstream of an EF- la promoter in the recombinant polynucleotide. In some embodiments, the sequence encoding human progranulin (e.g., hGRN sequence) is downstream of a putative MECP2 promoter in the recombinant polynucleotide. In some embodiments, the sequence encoding human progranulin (e.g., AGRA sequence) is downstream of a B-actin promoter in the recombinant polynucleotide. In some embodiments, the sequence encoding human progranulin (e.g., hGRN sequence) is downstream of a MND promoter in the recombinant polynucleotide.

Introns

[0154] In some embodiments, the recombinant polynucleotide comprises an intron. An intron can be a non-coding sequence present in the recombinant polynucleotide. In some embodiments, an intron is located within a protein coding sequence (e.g., within a sequence encoding progranulin). In some embodiments, an intron is located in an untranslated region of the polynucleotide (e.g., within a 5’ untranslated region). An intron can be removed during processing of the recombinant polynucleotide. For example, an intron can be removed from a sequence encoding progranulin prior to translation of the progranulin sequence. An intro can enhance expression of a payload encoded by the recombinant polynucleotide.

[0155] In some embodiments, an intron is a viral intron sequence (e.g., an SV40 intron). In some embodiments, an intron is a progranulin intron sequence (e.g., a human progranulin intron). For example, a recombinant polynucleotide can include one or more of human progranulin intron 1, human progranulin intron 2, human progranulin intron 3, human progranulin intron 4, human progranulin intron 5, human progranulin intron 6, human progranulin intron 7, human progranulin intron 8, human progranulin intron 9, human progranulin intron 10, human progranulin intron 11, or combinations thereof. An intron can be located in an endogenous position within a coding sequence. Exemplary sequences of introns are shown below in TABLE 3.

TABLE 3. Exemplary Sequences of Introns

[0156] In some embodiments, the recombinant polynucleotide further comprises a GRN intron. The GRN intron can comprise at least 80% sequence identity to any one of SEQ ID NO: 12 or SEQ ID NO: 27 - SEQ ID NO: 29. The GRN intron can comprise at least 85% sequence identity to any one of SEQ ID NO: 12 or SEQ ID NO: 27 - SEQ ID NO: 29. The GRN intron can comprise at least 90% sequence identity to any one of SEQ ID NO: 12 or SEQ ID NO: 27 - SEQ ID NO: 29. The GRN intron can comprise at least 95% sequence identity to any one of SEQ ID NO: 12 or SEQ ID NO: 27 - SEQ ID NO: 29. The GRN intron can comprise at least 96% sequence identity to any one of SEQ ID NO: 12 or SEQ ID NO: 27 - SEQ ID NO: 29. The GRN intron can comprise at least 97% sequence identity to any one of SEQ ID NO: 12 or SEQ ID NO: 27 - SEQ ID NO: 29. The GRN intron can comprise at least 98% sequence identity to any one of SEQ ID NO: 12 or SEQ ID NO: 27 - SEQ ID NO: 29. The GRN intron can comprise at least 99% sequence identity to any one of SEQ ID NO: 12 or SEQ ID NO: 27 - SEQ ID NO: 29. The GRN intron can comprise 100% sequence identity to any one of SEQ ID NO: 12 or SEQ ID NO: 27 - SEQ ID NO: 29. The recombinant polynucleotide can further comprise a GRN intron having any one of SEQ ID NO: 12 or SEQ ID NO: 27 - SEQ ID NO: 29. The GRN intron can be positioned within a 5’ untranslated region of the recombinant polynucleotide. The GRN intron can be positioned within a sequence encoding a payload (e.g., within a sequence encoding a progranulin payload). In some embodiments, the recombinant polynucleotide further comprises at least 1, 2, 3, 4, or 5 GRN introns.

[0157] For example, the recombinant polynucleotide further comprises a progranulin intron 9 (GRN intron 9). The GRN intron 9 can comprise at least 80% sequence identity to SEQ ID NO: 12. The GRN intron 9 can comprise at least 85% sequence identity to SEQ ID NO: 12. The GRN intron 9 can comprise at least 90% sequence identity to SEQ ID NO: 12. The GRN intron 9 can comprise at least 95% sequence identity to SEQ ID NO: 12. The GRN intron 9 can comprise at least 96% sequence identity to SEQ ID NO: 12. The GRN intron 9 can comprise at least 97% sequence identity to SEQ ID NO: 12. The GRN intron 9 can comprise at least 98% sequence identity to SEQ ID NO: 12. The GRN intron 9 can comprise at least 99% sequence identity to SEQ ID NO: 12. The GRN intron 9 can comprise 100% sequence identity to SEQ ID NO: 12. The recombinant polynucleotide can further comprise a GRN intron 9 having a sequence of SEQ ID NO: 12. The GRN intron 9 can be positioned within a 5’ untranslated region of the recombinant polynucleotide. The GRN intron 9 can be positioned within a sequence encoding a payload (e.g., within a sequence encoding a progranulin payload). In some embodiments, the recombinant polynucleotide further comprises at least 1, 2, 3, 4, or 5 GRN intron 9s.

[0158] In some embodiments, the recombinant polynucleotide further comprises an SV40 intron. The SV40 intron can comprise at least 80% sequence identity to SEQ ID NO: 13. The SV40 intron can comprise at least 85% sequence identity to SEQ ID NO: 13. The SV40 intron can comprise at least 90% sequence identity to SEQ ID NO: 13. The SV40 intron can comprise at least 95% sequence identity to SEQ ID NO: 13. The SV40 intron can comprise at least 96% sequence identity to SEQ ID NO: 13. The SV40 intron can comprise at least 97% sequence identity to SEQ ID NO: 13. The SV40 intron can comprise at least 98% sequence identity to SEQ ID NO: 13. The SV40 intron can comprise at least 99% sequence identity to SEQ ID NO: 13. The SV40 intron can comprise 100% sequence identity to SEQ ID NO: 13. The recombinant polynucleotide can further comprise an SV40 intron having a sequence of SEQ ID NO: 13. The SV40 intron can be positioned within a 5’ untranslated region of the recombinant polynucleotide. The SV40 intro can be positioned within a sequence encoding a payload (e.g., within a sequence encoding progranulin). In some embodiments, the recombinant polynucleotide further comprises at least 1, 2, 3, 4, or 5 SV40 introns.

[0159] In some embodiments, the recombinant polynucleotide further comprises an SV40 smT intron. The SV40 smT intron can comprise at least 80% sequence identity to SEQ ID NO: 30. The SV40 smT intron can comprise at least 85% sequence identity to SEQ ID NO: 30. The SV40 smT intron can comprise at least 90% sequence identity to SEQ ID NO: 30. The SV40 smT intron can comprise at least 95% sequence identity to SEQ ID NO: 30. The SV40 smT intron can comprise at least 96% sequence identity to SEQ ID NO: 30. The SV40 smT intron can comprise at least 97% sequence identity to SEQ ID NO: 30. The SV40 smT intron can comprise at least 98% sequence identity to SEQ ID NO: 30. The SV40 smT intron can comprise at least 99% sequence identity to SEQ ID NO: 30. The SV40 smT intron can comprise 100% sequence identity to SEQ ID NO: 30. The recombinant polynucleotide can further comprise an SV40 smT intron having a sequence of SEQ ID NO: 30. The SV40 smT intron can be positioned within a 5’ untranslated region of the recombinant polynucleotide. The SV40 smT intron can be positioned within a sequence encoding a payload (e.g., within a sequence encoding progranulin). In some embodiments, the recombinant polynucleotide further comprises at least 1, 2, 3, 4, or 5 SV40 smT introns.

[0160] In some embodiments, the recombinant polynucleotide further comprises a TPI intron 3. The TPI intron 3 can comprise at least 80% sequence identity to SEQ ID NO: 31. The TPI intron 3 can comprise at least 85% sequence identity to SEQ ID NO: 31. The TPI intron 3 can comprise at least 90% sequence identity to SEQ ID NO: 31. The TPI intron 3 can comprise at least 95% sequence identity to SEQ ID NO: 31. TPI intron 3 can comprise at least 96% sequence identity to SEQ ID NO: 31. The TPI intron 3 can comprise at least 97% sequence identity to SEQ ID NO: 31. The TPI intron 3 can comprise at least 98% sequence identity to SEQ ID NO: 31. The TPI intron 3 can comprise at least 99% sequence identity to SEQ ID NO: 31. The TPI intron 3 can comprise 100% sequence identity to SEQ ID NO: 31. The recombinant polynucleotide can further comprise a TPI intron 3 having a sequence of SEQ ID NO: 31. The TPI intron 3 can be positioned within a 5 ’ untranslated region of the recombinant polynucleotide. The TPI intron 3 can be positioned within a sequence encoding a payload (e.g., within a sequence encoding progranulin). In some embodiments, the recombinant polynucleotide further comprises at least 1, 2, 3, 4, or 5 SV40 TPI intron 3s.

Additional Elements

[0161] In some embodiments, the recombinant polynucleotide further comprises additional elements. In some embodiments, the additional elements can enhance or increase transcription or translation of the GRN sequence. The additional elements can enhance or increase expression of the payload, e.g., enhance or increase expression of progranulin. Examples of additional elements that can be included in a recombinant polynucleotide are a post-transcriptional regulatory element, a polyadenylation signal, a transcriptional pause site, a neuron-restrictive silencer element, a CCCTC-binding factor sequence, a 5’ untranslated region, a 3’ untranslated region, a 5’ stuffer sequence, and combinations thereof. For example, a recombinant polynucleotide comprises additional elements of a post-transcriptional regulatory element, a polyadenylation signal, and a transcriptional pause site. In a preferred example a recombinant polynucleotide comprises the following additional elements: a post- transcriptional regulatory element, a polyadenylation signal, a transcriptional pause site, a 5 ’ untranslated region, a 3 ’ untranslated region, and a 5’ stuffer sequence.

[0162] In some embodiments, an additional element is a post-transcriptional regulatory element, such as a woodchuck post-transcriptional regulatory element (WPRE). In preferred embodiments, the WPRE is a shortened WPRE, e.g., WPRE3.

[0163] In some embodiments, an additional element is a neuron-restrictive silencer element (NRSE). The NRSE can be positioned in the recombinant polynucleotide such that the NRSE regulates cell specificity of payload expression. For example, the NRSE can regulate cell specificity of GRN expression from a recombinant polynucleotide comprising an NRSE and a GRN coding sequence. The NRSE can enhance expression of a payload in neuronal cells, silence expression of the payload in non-neuronal cells, or a combination thereof. In some instances, the NRSE enhances expression of the payload in neuronal cells relative to other cell types by silencing expression of the payload in non-neuronal cells.

[0164] In preferred embodiments, an additional element is a polyadenylation (poly A) signal. The polyA signal sequence can regulate expression of the payload.

[0165] In some embodiments, an additional element is a CCCTC-binding factor (CTCF) sequence. The CTCF sequence can be positioned in the recombinant polynucleotide for payload expression such that the CTCF modifies accessibility of the recombinant polynucleotide to transcription machinery to enhance expression of the payload.

[0166] In preferred embodiments, an additional element is a 5’ untranslated region (5’ UTR).

The 5 ’ UTR can be positioned in the recombinant polynucleotide such that the 5 ’ UTR regulates payload expression.

[0167] In some embodiments, an additional element is a 3’ untranslated region (3’ UTR). The 3’ UTR can be positioned in the recombinant polynucleotide such that the 3 ’ UTR regulates payload expression. The polyA signal sequence can regulate expression of the payload.

[0168] In some embodiments, a recombinant polynucleotide comprises a combination of additional elements. For example, a recombinant nucleotide can comprise one or more additional elements selected from the group consisting of a WPRE, an NRSE, a CTCF, a polyA signal, a 5 ’ UTR, a 3 ’ UTR, and combinations thereof. Exemplary sequences of additional elements are shown below in TABLE 4.

TABLE 4. Exemplary Sequences of Additional Elements

[0169] In some embodiments, the recombinant polynucleotide further comprises a post- transcriptional regulatory element, such as a woodchuck hepatitis virus post-transcriptional regulatory element (“WPRE”, also referred to as a “WPRE element”). In some embodiments, a post-transcriptional regulatory element is included in a 3 ’untranslated region of a recombinant polynucleotide. A post-transcriptional regulatory element can enhance expression of a coding sequence (e.g., a sequence encoding a payload) of a recombinant polynucleotide.

[0170] The WPRE can comprise at least 80% sequence identity to SEQ ID NO: 7. The WPRE can comprise at least 85% sequence identity to SEQ ID NO: 7. The WPRE can comprise at least 90% sequence identity to SEQ ID NO: 7. The WPRE can comprise at least 95% sequence identity to SEQ ID NO: 7. The WPRE can comprise at least 96% sequence identity to SEQ ID NO: 7. The WPRE can comprise at least 97% sequence identity to SEQ ID NO: 7. The WPRE can comprise at least 98% sequence identity to SEQ ID NO: 7. The WPRE can comprise at least 99% sequence identity to SEQ ID NO: 7. The WPRE can comprise 100% sequence identity to SEQ ID NO: 7. The recombinant polynucleotide can further comprise a WPRE having a sequence of SEQ ID NO: 7. In some embodiments, the WPRE is downstream of the promoter in the recombinant polynucleotide. In some embodiments the WPRE is downstream of the sequence encoding the payload (e.g., the GRN sequence). In some embodiments, the recombinant polynucleotide further comprises at least 1, 2, 3, 4, or 5 WPREs. [0171] In preferred embodiments, the WPRE element is a WPRE variant. In some embodiments, the WPRE variant is a shortened WPRE or truncated WPRE. A shorted WPRE or truncated WPRE (e.g., a WPRE3) can exhibit similar transcriptional activity as a full length WPRE. A shorted WPRE or truncated WPRE can be preferred over a full length WPRE due to its shorter nucleotide sequence. In preferred embodiments, the shortened WPRE is a WPRE3. In preferred embodiments, the recombinant polynucleotide further comprises a WPRE3. The WPRE3 can comprise at least 80% sequence identity to SEQ ID NO: 8. The WPRE3 can comprise at least 85% sequence identity to SEQ ID NO: 8. The WPRE3 can comprise at least 90% sequence identity to SEQ ID NO: 8. The WPRE3 can comprise at least 95% sequence identity to SEQ ID NO: 8. The WPRE3 can comprise at least 96% sequence identity to SEQ ID NO: 8. The WPRE3 can comprise at least 97% sequence identity to SEQ ID NO: 8. The WPRE3 can comprise at least 98% sequence identity to SEQ ID NO: 8. The WPRE3 can comprise at least 99% sequence identity to SEQ ID NO: 8. The WPRE3 can comprise 100% sequence identity to SEQ ID NO: 8. The recombinant polynucleotide can further comprise a WPRE3 having a sequence of SEQ ID NO: 8. In some embodiments, the WPRE3 is downstream of the promoter in the recombinant polynucleotide. In some embodiments the WPRE3 is downstream of the sequence encoding the payload (e.g., the GRN sequence). In some embodiments, the recombinant polynucleotide further comprises at least 1, 2, 3, 4, or 5 WPRE3s.

[0172] In some embodiments, the post-transcriptional regulatory element is part of a 3’ untranslated region (3’ UTR). In some embodiments, a 3’ UTR comprises a post-transcriptional regulatory element.

[0173] In some embodiments, the recombinant polynucleotide further comprises a neuron restrictive silencer element (NRSE). A NRSE can inhibit expression of the payload sequence in non-neuronal cells. The NRSE can comprise at least 80% sequence identity to any one of SEQ ID NO: 32 - SEQ ID NO: 39. The NRSE can comprise at least 85% sequence identity to any one of SEQ ID NO: 32 - SEQ ID NO: 39. The NRSE can comprise at least 90% sequence identity to any one of SEQ ID NO: 32 - SEQ ID NO: 39. The NRSE can comprise at least 95% sequence identity to any one of SEQ ID NO: 32 - SEQ ID NO: 39. The NRSE can comprise at least 96% sequence identity to any one of SEQ ID NO: 32 - SEQ ID NO: 39. The NRSE can comprise at least 97% sequence identity to any one of SEQ ID NO: 32 - SEQ ID NO: 39. The NRSE can comprise at least 98% sequence identity to any one of SEQ ID NO: 32 - SEQ ID NO: 39. The NRSE can comprise at least 99% sequence identity to any one of SEQ ID NO: 32 - SEQ ID NO: 39. The NRSE can comprise 100% sequence identity to any one of SEQ ID NO: 32 - SEQ ID NO: 39. The recombinant polynucleotide can further comprise a NRSE having a sequence of any one of SEQ ID NO: 32 - SEQ ID NO: 39. In some embodiments, the NRSE is downstream of the promoter in the recombinant polynucleotide. In some embodiments the NRSE is downstream of the sequence encoding the payload (e.g., the GRN sequence). In some embodiments, the recombinant polynucleotide further comprises at least 1, 2, 3, 4, or 5 NRSEs. [0174] In some embodiments, the recombinant polynucleotide further comprises an NRSE and a CCCTC-binding factor (CTCF). The NRSE and CTCF can comprise at least 80% sequence identity to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 38. The NRSE and CTCF can comprise at least 85% sequence identity to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 38. The NRSE and CTCF can comprise at least 90% sequence identity to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 38. The NRSE and CTCF can comprise at least 95% sequence identity to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 38. The NRSE and CTCF can comprise at least 96% sequence identity to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 38. The NRSE and CTCF can comprise at least 97% sequence identity to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 38. The NRSE and CTCF can comprise at least 98% sequence identity to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 38. The NRSE and CTCF can comprise at least 99% sequence identity to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 38. The NRSE and CTCF can comprise 100% sequence identity to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 38. The recombinant polynucleotide can further comprise a NRSE and CTCF having a sequence of SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 38. In some embodiments, the NRSE and CTCF is downstream of the promoter in the recombinant polynucleotide. In some embodiments the NRSE and CTCF is downstream of the sequence encoding the payload (e.g., the GRN sequence). In some embodiments, the recombinant polynucleotide further comprises at least 1, 2, 3, 4, or 5 NRSEs and CTCFs.

[0175] In preferred embodiments, the recombinant polynucleotide further comprises a polyadenylation (poly A) signal. The polyA signal can signal formation of a polyA tail to an RNA transcribed from the recombinant polynucleotide. The polyA signal can comprise at least 80% sequence identity to any one of SEQ ID NO: 40 - SEQ ID NO: 48 or SEQ ID NO: 55. The polyA signal can comprise at least 85% sequence identity to any one of SEQ ID NO: 40 - SEQ ID NO: 48 or SEQ ID NO: 55. The polyA signal can comprise at least 90% sequence identity to any one of SEQ ID NO: 40 - SEQ ID NO: 48 or SEQ ID NO: 55. The polyA signal can comprise at least 95% sequence identity to any one of SEQ ID NO: 40 - SEQ ID NO: 48 or SEQ ID NO: 55. The polyA signal can comprise at least 96% sequence identity to any one of SEQ ID NO: 40 - SEQ ID NO: 48 or SEQ ID NO: 55. The polyA signal can comprise at least 97% sequence identity to any one of SEQ ID NO: 40 - SEQ ID NO: 48 or SEQ ID NO: 55. The polyA signal can comprise at least 98% sequence identity to any one of SEQ ID NO: 40 - SEQ ID NO: 48 or SEQ ID NO: 55. The polyA signal can comprise at least 99% sequence identity to any one of SEQ ID NO: 40 - SEQ ID NO: 48 or SEQ ID NO: 55. The polyA signal can comprise 100% sequence identity to any one of SEQ ID NO: 40 - SEQ ID NO: 48 or SEQ ID NO: 55. The recombinant polynucleotide can further comprise a polyA signal having a sequence of any one of SEQ ID NO: 40 - SEQ ID NO: 48 or SEQ ID NO: 55. In some embodiments, the polyA signal is downstream of the promoter in the recombinant polynucleotide. In some embodiments the polyA signal is downstream of the sequence encoding the payload (e.g., the GRN sequence). In some embodiments, the recombinant polynucleotide further comprises at least 1, 2, 3, 4, or 5 polyA signals.

[0176] In preferred embodiments, the polyA signal is a rabbit beta globin polyA signal. The rabbit beta globin rabbit beta globin polyA signal can comprise at least 80% sequence identity to SEQ ID NO: 42. The rabbit beta globin polyA signal can comprise at least 85% sequence identity to SEQ ID NO: 42. The rabbit beta globin polyA signal can comprise at least 90% sequence identity to SEQ ID NO: 42. The rabbit beta globin polyA signal can comprise at least 95% sequence identity to SEQ ID NO: 42. The rabbit beta globin polyA signal can comprise at least 96% sequence identity to SEQ ID NO: 42. The rabbit beta globin polyA signal can comprise at least 97% sequence identity to SEQ ID NO: 42. The rabbit beta globin polyA signal can comprise at least 98% sequence identity to SEQ ID NO: 42. The rabbit beta globin polyA signal can comprise at least 99% sequence identity to SEQ ID NO: 42. The rabbit beta globin polyA signal can comprise 100% sequence identity to SEQ ID NO: 42. The recombinant polynucleotide can further comprise a rabbit beta globin polyA signal having a sequence of SEQ ID NO: 42. In some embodiments, the rabbit beta globin polyA signal is downstream of the promoter in the recombinant polynucleotide. In some embodiments the rabbit beta globin polyA signal is downstream of the sequence encoding the payload (e.g., the GRN sequence). In some embodiments, the recombinant polynucleotide further comprises at least 1, 2, 3, 4, or 5 rabbit beta globin polyA signals.

[0177] The polyA signal can comprise at least 80% sequence identity to SEQ ID NO: 40 or SEQ ID NO: 41. The polyA signal can comprise at least 85% sequence identity to SEQ ID NO: 40 or SEQ ID NO: 41. The polyA signal can comprise at least 90% sequence identity to SEQ ID NO: 40 or SEQ ID NO: 41. The polyA signal can comprise at least 95% sequence identity to SEQ ID NO: 40 or SEQ ID NO: 41. The polyA signal can comprise at least 96% sequence identity to SEQ ID NO: 40 or SEQ ID NO: 41. The polyA signal can comprise at least 97% sequence identity to SEQ ID NO: 40 or SEQ ID NO: 41. The polyA signal can comprise at least 98% sequence identity to SEQ ID NO: 40 or SEQ ID NO: 41. The polyA signal can comprise at least 99% sequence identity to SEQ ID NO: 40 or SEQ ID NO: 41. The polyA signal can comprise 100% sequence identity to SEQ ID NO: 40 or SEQ ID NO: 41. The recombinant polynucleotide can further comprise a polyA signal having a sequence of SEQ ID NO: 40 or SEQ ID NO: 41. In some embodiments, the polyA signal is downstream of the promoter in the recombinant polynucleotide. In some embodiments the polyA signal is downstream of the sequence encoding the payload (e.g., the GRN sequence). In some embodiments, the recombinant polynucleotide further comprises at least 1, 2, 3, 4, or polyA signals.

[0178] In some embodiments, the polyA signal is part of a 3’ untranslated region (3’ UTR). In some embodiments, a 3’ UTR comprises a polyA signal.

[0179] In preferred embodiments, the recombinant polynucleotide further comprises a transcriptional pause site. The transcriptional pause site can facilitate transcriptional termination and prevent transcriptional interference. The transcriptional pause site can reduce transcriptional silencing, for example, transcriptional silencing caused by transcriptional readthrough from an upstream promoter. In some embodiments, the transcriptional pause site includes a second polyA signal. The transcriptional pause site can comprise at least 80% sequence identity to SEQ ID NO: 64. The transcriptional pause site can comprise at least 85% sequence identity to SEQ

ID NO: 64. The transcriptional pause site can comprise at least 90% sequence identity to SEQ

ID NO: 64. The transcriptional pause site can comprise at least 95% sequence identity to SEQ

ID NO: 64. The transcriptional pause site can comprise at least 96% sequence identity to SEQ

ID NO: 64. The transcriptional pause site can comprise at least 97% sequence identity to SEQ

ID NO: 64. The transcriptional pause site can comprise at least 98% sequence identity to SEQ

ID NO: 64. The transcriptional pause site can comprise at least 99% sequence identity to SEQ

ID NO: 64. The transcriptional pause site can comprise 100% sequence identity to SEQ ID NO: 64. The recombinant polynucleotide can further comprise a transcriptional pause site having a sequence of SEQ ID NO: 64. In some embodiments, the transcriptional pause site is downstream of the promoter in the recombinant polynucleotide. In some embodiments the transcriptional pause site is downstream of the sequence encoding the payload (e.g., the GRN sequence). In some embodiments, the transcriptional pause site is downstream of the polyadenylation signal. [0180] In preferred embodiments, the recombinant polynucleotide comprises a 5 ’ untranslated region (5 ’UTR), such as a minimal 5’ UTR. In some embodiments, a 5'UTR is from a transcriptional start site (TSS) to a translation initiation site (TIS) in a recombinant polynucleotide as disclosed herein. In some embodiments, the recombinant polynucleotide comprises a 5 ’ UTR containing an intron. In some embodiments, the recombinant polynucleotide comprises a 5 ’ UTR; an intron in the 5 ’ UTR, the sequence encoding the payload, or both; and a polyadenylation signal.

[0181] In some embodiments, the 5 ’UTR overlaps with the promoter. In some embodiments, the overlap is 33 nucleotides, wherein the 33 nucleotides are the 3’ 33 nucleotides of the promoter. The promoter can be an RSV promoter. The promoter can comprise SEQ ID NO: 3. The 5 ’UTR can comprise, from 5’ to 3’, the 3’ 33 nucleotides of SEQ ID NO: 3 and SEQ ID NO: 56. The 5’ UTR can comprise a Kozak sequence downstream of the promoter. The Kozak sequence can comprise SEQ ID NO: 66. The 5’ UTR can comprise, from 5’ to 3’, the 3’ 33 nucleotides of SEQ ID NO: 3 and SEQ ID NO: 66. The 5’ UTR can comprise additional nucleotides between the 3’ 33 nucleotides of the SEQ ID NO: 3 and SEQ ID NO: 66.

[0182] The 5’ UTR can comprise at least 80% sequence identity to SEQ ID NO: 62. The 5’ UTR can comprise at least 85% sequence identity to SEQ ID NO: 62. The 5’ UTR can comprise at least 90% sequence identity to SEQ ID NO: 62. The 5’ UTR can comprise at least 95% sequence identity to SEQ ID NO: 62. The 5’ UTR can comprise at least 96% sequence identity to SEQ ID NO: 62. The 5’ UTR can comprise at least 97% sequence identity to SEQ ID NO: 62. The 5’ UTR can comprise at least 98% sequence identity to SEQ ID NO: 62. The 5’ UTR can comprise at least 99% sequence identity to SEQ ID NO: 62. The 5’ UTR can comprise 100% sequence identity to SEQ ID NO: 62. In some embodiments, the 5’ UTR is downstream of the promoter in the recombinant polynucleotide. In some embodiments, the 5’ UTR overlaps the 3’ end of the promoter. In some embodiments the 5 ’ UTR is upstream of the sequence encoding the payload (e.g., the GRN sequence).

[0183] The 5’ UTR can comprise at least 80% sequence identity to SEQ ID NO: 56. The 5’ UTR can comprise at least 85% sequence identity to SEQ ID NO: 56. The 5’ UTR can comprise at least 90% sequence identity to SEQ ID NO: 56. The 5’ UTR can comprise at least 95% sequence identity to SEQ ID NO: 56. The 5’ UTR can comprise at least 96% sequence identity to SEQ ID NO: 56. The 5’ UTR can comprise at least 97% sequence identity to SEQ ID NO: 56. The 5’ UTR can comprise at least 98% sequence identity to SEQ ID NO: 56. The 5’ UTR can comprise at least 99% sequence identity to SEQ ID NO: 56. The 5’ UTR can comprise 100% sequence identity to SEQ ID NO: 56. In some embodiments, the 5’ UTR is downstream of the promoter in the recombinant polynucleotide. In some embodiments, the 5’ UTR overlaps the 3’ end of the promoter. In some embodiments the 5 ’ UTR is upstream of the sequence encoding the payload (e.g., the GRN sequence). [0184] The 5’ UTR can comprise a Kozak sequence. The Kozak sequence can comprise at least 80% sequence identity to SEQ ID NO: 66. The Kozak sequence can comprise at least 85% sequence identity to SEQ ID NO: 66. The Kozak sequence can comprise at least 90% sequence identity to SEQ ID NO: 66. The Kozak sequence can comprise at least 95% sequence identity to SEQ ID NO: 66. The Kozak sequence can comprise at least 96% sequence identity to SEQ ID NO: 66. The Kozak sequence can comprise at least 97% sequence identity to SEQ ID NO: 66. The Kozak sequence can comprise at least 98% sequence identity to SEQ ID NO: 66. The Kozak sequence can comprise at least 99% sequence identity to SEQ ID NO: 66. The Kozak sequence can comprise 100% sequence identity to SEQ ID NO: 66. In some embodiments, the Kozak sequence is downstream of the promoter in the recombinant polynucleotide. In some embodiments the Kozak sequence is upstream of the sequence encoding the payload (e.g., the GRN sequence).

[0185] In some embodiments, the recombinant polynucleotide further comprises a stuffer sequence. The stuffer sequence can comprise at least 80% sequence identity to SEQ ID NO: 61. The stuffer sequence can comprise at least 85% sequence identity to SEQ ID NO: 61. The stuffer sequence can comprise at least 90% sequence identity to SEQ ID NO: 61. The stuffer sequence can comprise at least 95% sequence identity to SEQ ID NO: 61. The stuffer sequence can comprise at least 96% sequence identity to SEQ ID NO: 61. The stuffer sequence can comprise at least 97% sequence identity to SEQ ID NO: 61. The stuffer sequence can comprise at least 98% sequence identity to SEQ ID NO: 61. The stuffer sequence can comprise at least 99% sequence identity to SEQ ID NO: 61. The stuffer sequence can comprise 100% sequence identity to SEQ ID NO: 61. The recombinant polynucleotide can further comprise a stuffer sequence having a sequence of SEQ ID NO: 61. In some embodiments, the stuffer sequence is upstream of the promoter in the recombinant polynucleotide. In some embodiments the stuffer sequence is upstream of the sequence encoding the payload (e.g., the GRN sequence). In some embodiments, the recombinant polynucleotide further comprises at least 1, 2, 3, 4, or stuffer sequences.

[0186] In preferred embodiments, the recombinant polynucleotide further comprises a 5’ inverted terminal repeat (5’ ITR). The 5’ ITR can be selected based on a vector, such as a viral vector, used to deliver the recombinant polynucleotide. In some embodiments, the 5’ ITR can be an AAV 5’ ITR (e.g., an AAV5 5’ ITR, an AAV1 5’ ITR, an AAV2 5’ ITR, an AAV9 5’ ITR, or a PhP.eB 5’ ITR). The 5’ ITR can be a single stranded AAV 5’ ITR (e.g., a single stranded AAV2 5’ ITR). The 5’ ITR can be a self-complementary AAV 5’ ITR (e.g., a self- complementary AAV2 5’ ITR). The 5’ ITR can comprise at least 80% sequence identity to SEQ ID NO: 59. The 5’ ITR can comprise at least 85% sequence identity to SEQ ID NO: 59. The 5’ ITR can comprise at least 90% sequence identity to SEQ ID NO: 59. The 5’ ITR can comprise at least 95% sequence identity to SEQ ID NO: 59. The 5’ ITR can comprise at least 96% sequence identity to SEQ ID NO: 59. The 5’ ITR can comprise at least 97% sequence identity to SEQ ID NO: 59. The 5’ ITR can comprise at least 98% sequence identity to SEQ ID NO: 59. The 5’ ITR can comprise at least 99% sequence identity to SEQ ID NO: 59. The 5’ ITR can comprise 100% sequence identity to SEQ ID NO: 59. In some embodiments, the 5’ ITR is upstream of the promoter in the recombinant polynucleotide. In some embodiments the 5’ ITR is upstream of the sequence encoding the payload (e.g., the GRN sequence). In some embodiments, the recombinant polynucleotide comprises at least 1, 2, 3, 4, or 5’ ITRs.

[0187] In preferred embodiments, the recombinant polynucleotide further comprises a 3’ inverted terminal repeat (3’ ITR). The 3’ ITR can be selected based on a vector used to deliver the recombinant polynucleotide. In some embodiments, the 3’ ITR can be an AAV 3’ ITR (e.g., an AAV5 3’ ITR, an AAV1 3’ ITR, an AAV2 3’ ITR, an AAV9 3’ ITR, or a PhP.eB 3’ ITR). The 3’ ITR can be a single stranded AAV 3’ ITR (e.g., a single stranded AAV2 3’ ITR). The 3’ ITR can be a self-complementary AAV 3’ ITR (e.g., a self-complementary AAV2 3’ ITR). The 3’ ITR can comprise at least 80% sequence identity to SEQ ID NO: 60. The 3’ ITR can comprise at least 85% sequence identity to SEQ ID NO: 60. The 3’ ITR can comprise at least 90% sequence identity to SEQ ID NO: 60. The 3’ ITR can comprise at least 95% sequence identity to SEQ ID NO: 60. The 3’ ITR can comprise at least 96% sequence identity to SEQ ID NO: 60. The 3’ ITR can comprise at least 97% sequence identity to SEQ ID NO: 60. The 3’ ITR can comprise at least 98% sequence identity to SEQ ID NO: 60. The 3’ ITR can comprise at least 99% sequence identity to SEQ ID NO: 60. The 3’ ITR can comprise 100% sequence identity to SEQ ID NO: 60. In some embodiments, the 3’ ITR is downstream of the promoter in the recombinant polynucleotide. In some embodiments the 3’ ITR is downstream of the sequence encoding the payload (e.g., the GRN sequence). In some embodiments, the recombinant polynucleotide comprises at least 1, 2, 3, 4, or 3’ ITRs.

[0188] In some embodiments, the coding sequence (e.g., the GRN sequence) is downstream of the RSV promoter, the post-transcriptional regulatory element is downstream of the coding sequence, the polyadenylation signal is downstream of the post-transcriptional regulatory element, and the transcriptional pause site is downstream of the polyadenylation signal. In some embodiments, the stuffer sequence is upstream of the RSV promoter, the coding sequence is downstream of the RSV promoter, the post-transcriptional regulatory element is downstream of the coding sequence (e.g., the GRN sequence), the polyadenylation signal is downstream of the post-transcriptional regulatory element, and the transcriptional pause site is downstream of the polyadenylation signal. In some embodiments, the stuffer sequence is upstream of the RSV promoter, the 5 ’ untranslated region is downstream of the RSV promoter, the coding sequence (e.g., the GRN sequence) is downstream of the 5’ untranslated region, the post-transcriptional regulatory element is downstream of the coding sequence, the polyadenylation signal is downstream of the post-transcriptional regulatory element, and the transcriptional pause site is downstream of the polyadenylation signal. In some embodiments, the 5’ inverted terminal repeat is upstream of the stuffer sequence and the 3 ’ inverted terminal repeat downstream of the transcriptional pause site. In some embodiments, wherein the 5 ’ inverted terminal repeat is upstream of the stuffer sequence, the stuffer sequence is upstream of the RSV promoter, the 5 ’ untranslated region is downstream of the RSV promoter, the coding sequence is downstream of the 5 ’ untranslated region, the post-transcriptional regulatory element is downstream of the coding sequence, the polyadenylation signal is downstream of the post-transcriptional regulatory element, the transcriptional pause site is downstream of the polyadenylation signal, and the 3 ’ inverted terminal repeat downstream of the transcriptional pause site.

[0189] In some embodiments, a recombinant polynucleotide includes one or more cloning sites. A cloning site can include a restriction site that can be specifically cut by a restriction enzyme. In some embodiments, a cloning site is a multiple cloning site comprising multiple restriction sites (e.g., from 2 to 20 restriction sites). A cloning site can be included between sequence elements within a recombinant polynucleotide (e.g., between a coding sequence and an additional element, between a coding sequence and a promoter, between a promoter and an additional element, between two additional elements, or combinations thereof). A cloning site can comprise a restriction enzyme cut site (also referred to as a “cut site” or a “restriction site”). Examples of cloning sites that can be included in a recombinant polynucleotide of the present disclosure are a Hindlll cut site, a Xball cut site, a Notl cut site, a Sall cut site, a PstI cut site, and combinations thereof. In some embodiments, a recombinant polynucleotide includes a cloning site between a progranulin coding sequence and a post-transcriptional regulatory element. For example, a recombinant polynucleotide includes a Hindlll cut site between a progranulin coding sequence (e.g., SEQ ID NO: 10) and a WPRE3 post-transcriptional regulatory element (e.g., SEQ ID NO: 8). In some embodiments, a recombinant polynucleotide includes a cloning site between a post-transcriptional regulatory element and a polyadenylation site. For example, a recombinant polynucleotide includes a Xball cut site between a WPRE3 post-transcriptional regulatory element (e.g., SEQ ID NO: 8) and a rabbit beta globin polyadenylation site (e.g., SEQ ID NO: 42). In some embodiments, a recombinant polynucleotide includes a cloning site between a polyadenylation site and a transcriptional pause site. For example, a recombinant polynucleotide includes a Notl cut site between a rabbit beta globin polyadenylation site (e.g., SEQ ID NO: 42) and a transcriptional pause site (e.g., SEQ ID NO: 64). In some embodiments, a recombinant polynucleotide includes a cloning site between a transcriptional pause site and a 3’ inverted terminal repeat. For example, a recombinant polynucleotide includes a Sall cut site and a PstI cut site between a transcriptional pause site (e.g., SEQ ID NO: 64) and a 3’ inverted terminal repeat (e.g., SEQ ID NO: 60). In some embodiments, a recombinant polynucleotide includes a cloning site between a progranulin coding sequence and a post-transcriptional regulatory element, a cloning site between a post- transcriptional regulatory element and a polyadenylation site, a cloning site between a polyadenylation site and a transcriptional pause site, and a cloning site between a transcriptional pause site and a 3’ inverted terminal repeat. For example, a recombinant polynucleotide includes a Hindlll cut site between a progranulin coding sequence (e.g., SEQ ID NO: 10) and a WPRE3 post-transcriptional regulatory element (e.g., SEQ ID NO: 8), a Xball cut site between a WPRE3 post-transcriptional regulatory element (e.g., SEQ ID NO: 8) and a rabbit beta globin polyadenylation site (e.g., SEQ ID NO: 42), a Notl cut site between a rabbit beta globin polyadenylation site (e.g., SEQ ID NO: 42) and a transcriptional pause site (e.g., SEQ ID NO: 64), and a Sall cut site and a PstI cut site between a transcriptional pause site (e.g., SEQ ID NO: 64) and a 3’ inverted terminal repeat (e.g., SEQ ID NO: 60).

Recombinant Polynucleotides Encoding Progranulin

[0190] A recombinant polynucleotide can comprise a coding sequence encoding a progranulin payload operably linked to a promoter (e.g., an RSV promoter such as SEQ ID NO: 3). The recombinant polynucleotide can encode for expression of progranulin. The recombinant polynucleotide can include one or more additional elements. For example, the recombinant polynucleotide comprises a post-transcriptional regulatory element (e.g., a WPRE3 of SEQ ID NO: 8), a polyadenylation signal (e.g., a polyadenylation signal of SEQ ID NO: 42), and a transcriptional pause site (e.g., a transcriptional pause site of SEQ ID NO: 64). Examples of recombinant polynucleotide sequences encoding progranulin are provided in TABLE 5.

TABLE 5. Exemplary Recombinant Polynucleotide Sequences

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[0191] In a preferred embodiment, the recombinant polynucleotide comprises an RSV promoter (e.g., SEQ ID NO: 3), a coding sequence encoding a progranulin protein (e.g., encoding a protein of SEQ ID NO: 63), a post-transcriptional regulator element (e.g., a WPRE3 of SEQ ID NO: 8), and a transcriptional pause site (e.g., SEQ ID NO: 64). In some embodiments, the recombinant polynucleotide further comprises a 5’ stuffer sequence (e.g., SEQ ID NO: 61). In some embodiments, the recombinant polynucleotide comprises a 5’ untranslated region. The 5’ untranslated region can comprise a sequence that overlaps with the promoter (e.g., SEQ ID NO: 62 which includes the 3’ 33 nucleotides of the RSV promoter). The 5’ untranslated region can comprise a Kozak sequence (e.g., SEQ ID NO: 66) and can further comprise additional nucleotides 5’ of the Kozak sequence (e.g., comprising 33 nucleotides overlapping the 3’ end of the promoter and comprising additional nucleotides between the 3’ end of the promoter and the Kozak sequence). The 5’ untranslated region can comprise a sequence of SEQ ID NO: 56 and can further comprise a region 5’ of SEQ ID NO: 56 that overlaps the promoter (e.g., comprising 33 nucleotides overlapping the 3’ end of the promoter). For example, the recombinant polynucleotide comprises a sequence of SEQ ID NO: 65.

[0192] In some embodiments, a recombinant polynucleotide (e.g., a preferred recombinant polynucleotide of SEQ ID NO: 121 or 122) can comprise, from 5’ to 3’, a 5’ stuffer sequence (e.g., SEQ ID NO: 61), an RSV promoter (e.g., SEQ ID NO: 3), a 5’ UTR (e.g., SEQ ID NO: 62 which overlaps with the RSV promoter and comprises 33 nucleotides of the 3’ end of the RSV promoter and further comprises a Kozak sequence of SEQ ID NO: 66), a coding sequence (e.g., SEQ ID NO: 10 comprising a sequence of SEQ ID NO: 57 encoding a progranulin signal peptide and a sequence of SEQ ID NO: 58), a post-transcriptional regulatory element (e.g., SEQ ID NO: 8), a polyA signal (e.g., SEQ ID NO: 42), and a transcriptional pause site (e.g., SEQ ID NO: 64).

[0193] The recombinant polynucleotide can comprise at least 80% sequence identity to SEQ ID NO: 121. The recombinant polynucleotide can comprise at least 85% sequence identity to SEQ ID NO: 121. The recombinant polynucleotide can comprise at least 90% sequence identity to SEQ ID NO: 121. The recombinant polynucleotide can comprise at least 95% sequence identity to SEQ ID NO: 121. The recombinant polynucleotide can comprise at least 96% sequence identity to SEQ ID NO: 121. The recombinant polynucleotide can comprise at least 97% sequence identity to SEQ ID NO: 121. The recombinant polynucleotide can comprise at least 98% sequence identity to SEQ ID NO: 121. The recombinant polynucleotide can comprise at least 99% sequence identity to SEQ ID NO: 121. The recombinant polynucleotide can comprise 100% sequence identity to SEQ ID NO: 121. The recombinant polynucleotide can comprise a sequence of SEQ ID NO: 121.

[0194] The recombinant polynucleotide can comprise at least 80% sequence identity to SEQ ID NO: 122. The recombinant polynucleotide can comprise at least 85% sequence identity to SEQ ID NO: 122. The recombinant polynucleotide can comprise at least 90% sequence identity to SEQ ID NO: 122. The recombinant polynucleotide can comprise at least 95% sequence identity to SEQ ID NO: 122. The recombinant polynucleotide can comprise at least 96% sequence identity to SEQ ID NO: 122. The recombinant polynucleotide can comprise at least 97% sequence identity to SEQ ID NO: 122. The recombinant polynucleotide can comprise at least 98% sequence identity to SEQ ID NO: 122. The recombinant polynucleotide can comprise at least 99% sequence identity to SEQ ID NO: 122. The recombinant polynucleotide can comprise 100% sequence identity to SEQ ID NO: 122. The recombinant polynucleotide can comprise a sequence of SEQ ID NO: 122.

[0195] In some embodiments, a recombinant polynucleotide (e.g., a preferred recombinant polynucleotide of SEQ ID NO: 123 or 124) can comprise, from 5’ to 3’, an RSV promoter (e.g., SEQ ID NO: 3), a 5’ UTR (e.g., SEQ ID NO: 62 which overlaps with the RSV promoter and comprises 33 nucleotides of the 3’ end of the RSV promoter and further comprises a Kozak sequence of SEQ ID NO: 66), a coding sequence (e.g., SEQ ID NO: 10 comprising a sequence of SEQ ID NO: 57 encoding a progranulin signal peptide and a sequence of SEQ ID NO: 58), a post-transcriptional regulatory element (e.g., SEQ ID NO: 8), a polyA signal (e.g., SEQ ID NO: 42), and a transcriptional pause site (e.g., SEQ ID NO: 64).

[0196] In a preferred embodiment, a recombinant polynucleotide (e.g., a preferred recombinant polynucleotide of SEQ ID NO: 65, as shown in FIG. 26) can comprise, from 5’ to 3’, a 5’ ITR (e.g., SEQ ID NO: 59), a 5’ stuffer sequence (e.g., SEQ ID NO: 61), an RSV promoter (e.g., SEQ ID NO: 3), a 5’ UTR (e.g., SEQ ID NO: 62 which overlaps with the RSV promoter and comprises 33 nucleotides of the 3’ end of the RSV promoter and further comprises a Kozak sequence of SEQ ID NO: 66), a coding sequence (e.g., SEQ ID NO: 10 comprising a sequence of SEQ ID NO: 57 encoding a progranulin signal peptide and a sequence of SEQ ID NO: 58), a post-transcriptional regulatory element (e.g., SEQ ID NO: 8), a polyA signal (e.g., SEQ ID NO: 42), a transcriptional pause site (e.g., SEQ ID NO: 64), and a 3’ ITR (e.g., SEQ ID NO: 60).

[0197] The recombinant polynucleotide can comprise at least 80% sequence identity to SEQ ID NO: 123. The recombinant polynucleotide can comprise at least 85% sequence identity to SEQ ID NO: 123. The recombinant polynucleotide can comprise at least 90% sequence identity to SEQ ID NO: 123. The recombinant polynucleotide can comprise at least 95% sequence identity to SEQ ID NO: 123. The recombinant polynucleotide can comprise at least 96% sequence identity to SEQ ID NO: 123. The recombinant polynucleotide can comprise at least 97% sequence identity to SEQ ID NO: 123. The recombinant polynucleotide can comprise at least 98% sequence identity to SEQ ID NO: 123. The recombinant polynucleotide can comprise at least 99% sequence identity to SEQ ID NO: 123. The recombinant polynucleotide can comprise 100% sequence identity to SEQ ID NO: 123. The recombinant polynucleotide can comprise a sequence of SEQ ID NO: 123.

[0198] The recombinant polynucleotide can comprise at least 80% sequence identity to SEQ ID NO: 124. The recombinant polynucleotide can comprise at least 85% sequence identity to SEQ ID NO: 124. The recombinant polynucleotide can comprise at least 90% sequence identity to SEQ ID NO: 124. The recombinant polynucleotide can comprise at least 95% sequence identity to SEQ ID NO: 124. The recombinant polynucleotide can comprise at least 96% sequence identity to SEQ ID NO: 124. The recombinant polynucleotide can comprise at least 97% sequence identity to SEQ ID NO: 124. The recombinant polynucleotide can comprise at least 98% sequence identity to SEQ ID NO: 124. The recombinant polynucleotide can comprise at least 99% sequence identity to SEQ ID NO: 124. The recombinant polynucleotide can comprise 100% sequence identity to SEQ ID NO: 124. The recombinant polynucleotide can comprise a sequence of SEQ ID NO: 124. [0199] The recombinant polynucleotide can comprise at least 80% sequence identity to SEQ ID NO: 65. The recombinant polynucleotide can comprise at least 85% sequence identity to SEQ ID NO: 65. The recombinant polynucleotide can comprise at least 90% sequence identity to SEQ ID NO: 65. The recombinant polynucleotide can comprise at least 95% sequence identity to SEQ ID NO: 65. The recombinant polynucleotide can comprise at least 96% sequence identity to SEQ ID NO: 65. The recombinant polynucleotide can comprise at least 97% sequence identity to SEQ ID NO: 65. The recombinant polynucleotide can comprise at least 98% sequence identity to SEQ ID NO: 65. The recombinant polynucleotide can comprise at least 99% sequence identity to SEQ ID NO: 65. The recombinant polynucleotide can comprise 100% sequence identity to SEQ ID NO: 65. The recombinant polynucleotide can comprise a sequence of SEQ ID NO: 65.

[0200] The recombinant polynucleotide can comprise at least 80% sequence identity to SEQ ID NO: 14. The recombinant polynucleotide can comprise at least 85% sequence identity to SEQ ID NO: 14. The recombinant polynucleotide can comprise at least 90% sequence identity to SEQ ID NO: 14. The recombinant polynucleotide can comprise at least 95% sequence identity to SEQ ID NO: 14. The recombinant polynucleotide can comprise at least 96% sequence identity to SEQ ID NO: 14. The recombinant polynucleotide can comprise at least 97% sequence identity to SEQ ID NO: 14. The recombinant polynucleotide can comprise at least 98% sequence identity to SEQ ID NO: 14. The recombinant polynucleotide can comprise at least 99% sequence identity to SEQ ID NO: 14. The recombinant polynucleotide can comprise 100% sequence identity to SEQ ID NO: 14. The recombinant polynucleotide can comprise a sequence of SEQ ID NO: 14.

[0201] The recombinant polynucleotide can comprise at least 80% sequence identity to SEQ ID NO: 15. The recombinant polynucleotide can comprise at least 85% sequence identity to SEQ ID NO: 15. The recombinant polynucleotide can comprise at least 90% sequence identity to SEQ ID NO: 15. The recombinant polynucleotide can comprise at least 95% sequence identity to SEQ ID NO: 15. The recombinant polynucleotide can comprise at least 96% sequence identity to SEQ ID NO: 15. The recombinant polynucleotide can comprise at least 97% sequence identity to SEQ ID NO: 15. The recombinant polynucleotide can comprise at least 98% sequence identity to SEQ ID NO: 15. The recombinant polynucleotide can comprise at least 99% sequence identity to SEQ ID NO: 15. The recombinant polynucleotide can comprise 100% sequence identity to SEQ ID NO: 15. The recombinant polynucleotide can comprise a sequence of SEQ ID NO: 15. [0202] The recombinant polynucleotide can comprise at least 80% sequence identity to SEQ ID NO: 16. The recombinant polynucleotide can comprise at least 85% sequence identity to SEQ ID NO: 16. The recombinant polynucleotide can comprise at least 90% sequence identity to SEQ ID NO: 16. The recombinant polynucleotide can comprise at least 95% sequence identity to SEQ ID NO: 16. The recombinant polynucleotide can comprise at least 96% sequence identity to SEQ ID NO: 16. The recombinant polynucleotide can comprise at least 97% sequence identity to SEQ ID NO: 16. The recombinant polynucleotide can comprise at least 98% sequence identity to SEQ ID NO: 16. The recombinant polynucleotide can comprise at least 99% sequence identity to SEQ ID NO: 16. The recombinant polynucleotide can comprise 100% sequence identity to SEQ ID NO: 16. The recombinant polynucleotide can comprise a sequence of SEQ ID NO: 16.

[0203] The recombinant polynucleotide can comprise at least 80% sequence identity to SEQ ID NO: 17. The recombinant polynucleotide can comprise at least 85% sequence identity to SEQ ID NO: 17. The recombinant polynucleotide can comprise at least 90% sequence identity to SEQ ID NO: 17. The recombinant polynucleotide can comprise at least 95% sequence identity to SEQ ID NO: 17. The recombinant polynucleotide can comprise at least 96% sequence identity to SEQ ID NO: 17. The recombinant polynucleotide can comprise at least 97% sequence identity to SEQ ID NO: 17. The recombinant polynucleotide can comprise at least 98% sequence identity to SEQ ID NO: 17. The recombinant polynucleotide can comprise at least 99% sequence identity to SEQ ID NO: 17. The recombinant polynucleotide can comprise 100% sequence identity to SEQ ID NO: 17. The recombinant polynucleotide can comprise a sequence of SEQ ID NO: 17.

[0204] The recombinant polynucleotide can comprise at least 80% sequence identity to SEQ ID NO: 18. The recombinant polynucleotide can comprise at least 85% sequence identity to SEQ ID NO: 18. The recombinant polynucleotide can comprise at least 90% sequence identity to SEQ ID NO: 18. The recombinant polynucleotide can comprise at least 95% sequence identity to SEQ ID NO: 18. The recombinant polynucleotide can comprise at least 96% sequence identity to SEQ ID NO: 18. The recombinant polynucleotide can comprise at least 97% sequence identity to SEQ ID NO: 18. The recombinant polynucleotide can comprise at least 98% sequence identity to SEQ ID NO: 18. The recombinant polynucleotide can comprise at least 99% sequence identity to SEQ ID NO: 18. The recombinant polynucleotide can comprise 100% sequence identity to SEQ ID NO: 18. The recombinant polynucleotide can comprise a sequence of SEQ ID NO: 18. [0205] The recombinant polynucleotide can comprise at least 80% sequence identity to SEQ ID NO: 67. The recombinant polynucleotide can comprise at least 85% sequence identity to SEQ ID NO: 67. The recombinant polynucleotide can comprise at least 90% sequence identity to SEQ ID NO: 67. The recombinant polynucleotide can comprise at least 95% sequence identity to SEQ ID NO: 67. The recombinant polynucleotide can comprise at least 96% sequence identity to SEQ ID NO: 67. The recombinant polynucleotide can comprise at least 97% sequence identity to SEQ ID NO: 67. The recombinant polynucleotide can comprise at least 98% sequence identity to SEQ ID NO: 67. The recombinant polynucleotide can comprise at least 99% sequence identity to SEQ ID NO: 67. The recombinant polynucleotide can comprise 100% sequence identity to SEQ ID NO: 67. The recombinant polynucleotide can comprise a sequence of SEQ ID NO: 67.

[0206] The recombinant polynucleotide can comprise at least 80% sequence identity to SEQ ID NO: 68. The recombinant polynucleotide can comprise at least 85% sequence identity to SEQ ID NO: 68. The recombinant polynucleotide can comprise at least 90% sequence identity to SEQ ID NO: 68. The recombinant polynucleotide can comprise at least 95% sequence identity to SEQ ID NO: 68. The recombinant polynucleotide can comprise at least 96% sequence identity to SEQ ID NO: 68. The recombinant polynucleotide can comprise at least 97% sequence identity to SEQ ID NO: 68. The recombinant polynucleotide can comprise at least 98% sequence identity to SEQ ID NO: 68. The recombinant polynucleotide can comprise at least 99% sequence identity to SEQ ID NO: 68. The recombinant polynucleotide can comprise 100% sequence identity to SEQ ID NO: 68. The recombinant polynucleotide can comprise a sequence of SEQ ID NO: 68.

[0207] The recombinant polynucleotide can comprise at least 80% sequence identity to SEQ ID NO: 69. The recombinant polynucleotide can comprise at least 85% sequence identity to SEQ ID NO: 69. The recombinant polynucleotide can comprise at least 90% sequence identity to SEQ ID NO: 69. The recombinant polynucleotide can comprise at least 95% sequence identity to SEQ ID NO: 69. The recombinant polynucleotide can comprise at least 96% sequence identity to SEQ ID NO: 69. The recombinant polynucleotide can comprise at least 97% sequence identity to SEQ ID NO: 69. The recombinant polynucleotide can comprise at least 98% sequence identity to SEQ ID NO: 69. The recombinant polynucleotide can comprise at least 99% sequence identity to SEQ ID NO: 69. The recombinant polynucleotide can comprise 100% sequence identity to SEQ ID NO: 69. The recombinant polynucleotide can comprise a sequence of SEQ ID NO: 69. [0208] The recombinant polynucleotide can comprise at least 80% sequence identity to SEQ ID NO: 70. The recombinant polynucleotide can comprise at least 85% sequence identity to SEQ ID NO: 70. The recombinant polynucleotide can comprise at least 90% sequence identity to SEQ ID NO: 70. The recombinant polynucleotide can comprise at least 95% sequence identity to SEQ ID NO: 70. The recombinant polynucleotide can comprise at least 96% sequence identity to SEQ ID NO: 70. The recombinant polynucleotide can comprise at least 97% sequence identity to SEQ ID NO: 70. The recombinant polynucleotide can comprise at least 98% sequence identity to SEQ ID NO: 70. The recombinant polynucleotide can comprise at least 99% sequence identity to SEQ ID NO: 70. The recombinant polynucleotide can comprise 100% sequence identity to SEQ ID NO: 70. The recombinant polynucleotide can comprise a sequence of SEQ ID NO: 70.

Therapeutic Applications

[0209] A recombinant polynucleotide, plasmids, viral vectors, and pharmaceutical compositions of the present disclosure can have therapeutic applications. A recombinant polynucleotide of the present disclosure encoding a payload under transcriptional control of a promoter can have therapeutic applications. Therapeutic applications include treatment of a disease or disorder. Treatment of a disease or disorder can comprise preventing, alleviating a symptom of, reversing progression of, or reducing an underlying cause of the disease or condition. The recombinant polynucleotides described herein can facilitate the therapeutic use by allowing for payload expression at a high enough level for the payload to have a therapeutic effect.

[0210] For example, a recombinant polynucleotide of the present disclosure encoding a payload under transcriptional control of a promoter express a peptide encoded by a payload sequence. The payload sequence can encode a peptide associated with a disease. For example, the payload sequence encodes a functional copy of a peptide to treat a disease associated with a mutation in or decreased expression of the peptide. Expression of the peptide associated with the disease can treat the disease. In some embodiments, the payload encodes peptide fragment. In some embodiments, the payload sequence is a GRN sequence that is used to treat FTD. Treatment of FTD can comprise expressing progranulin in a subject. In some embodiments, treatment of FTD comprises expressing progranulin in a fluid (e.g., a cerebrospinal fluid) of a subject. The fluid can be secreted by a cell or tissue expressing the progranulin. The progranulin can be expressed in the cerebrospinal fluid at a level high enough to have a therapeutic effect. In some embodiments, the progranulin level high enough to have a therapeutic effect is at least 3 ng/mL.

In some embodiments, the progranulin level to have a therapeutic effect is from 1 ng/mL to 3 ng/mL. In some embodiments, the progranulin level to have a therapeutic effect is from 1 ng/mL to 4 ng/mL. In some embodiments, the progranulin level to have a therapeutic effect is from 2 ng/mL to 4.5 ng/mL. In some embodiments, the progranulin level to have a therapeutic effect is from 1 ng/mL to 10 ng/mL. In some embodiments, a progranulin level in a serum of the subject is not more than 300 ng/mL.

[0211] In some embodiments, a recombinant polynucleotide of the present disclosure encoding GRN can be used to treat Amyotrophic Lateral Sclerosis (ALS). Treatment of ALS can comprise expressing progranulin in a subject. In some embodiments, treatment of ALS comprises expressing progranulin in a cerebrospinal fluid of a subject. The progranulin can be expressed in the cerebrospinal fluid at a level high enough to have a therapeutic effect. In some embodiments, the progranulin level high enough to have a therapeutic effect is at least 3 ng/mL. In some embodiments, the progranulin level to have a therapeutic effect is from 1 ng/mL to 3 ng/mL. In some embodiments, the progranulin level to have a therapeutic effect is from 2 ng/mL to 4.5 ng/mL. In some embodiments, the progranulin level to have a therapeutic effect is from 1 ng/mL to 10 ng/mL. In some embodiments, a progranulin level in a serum of the subject is not more than 300 ng/mL.

[0212] In some embodiments, a recombinant polynucleotide of the present disclosure encoding GRN can be used to treat Alzheimer’s Disease. Treatment of Alzheimer’s Disease can comprise expressing progranulin in a subject. In some embodiments, treatment of Alzheimer’s Disease comprises expressing progranulin in a cerebrospinal fluid of a subject. The progranulin can be expressed in the cerebrospinal fluid at a level high enough to have a therapeutic effect. In some embodiments, the progranulin level high enough to have a therapeutic effect is at least 3 ng/mL. In some embodiments, the progranulin level to have a therapeutic effect is from 1 ng/mL to 3 ng/mL. In some embodiments, the progranulin level to have a therapeutic effect is from 2 ng/mL to 4.5 ng/mL. In some embodiments, the progranulin level to have a therapeutic effect is from 1 ng/mL to 10 ng/mL. In some embodiments, a progranulin level in a serum of the subject is not more than 300 ng/mL.

[0213] In some embodiments, a recombinant polynucleotide of the present disclosure encoding GRN can be used to treat Parkinson’s Disease. Treatment of Parkinson’s Disease can comprise expressing progranulin in a subject. In some embodiments, treatment of Parkinson’s Disease comprises expressing progranulin in a cerebrospinal fluid of a subject. The progranulin can be expressed in the cerebrospinal fluid at a level high enough to have a therapeutic effect. In some embodiments, the progranulin level high enough to have a therapeutic effect is at least 3 ng/mL. In some embodiments, the progranulin level to have a therapeutic effect is from 1 ng/mL to 3 ng/mL. In some embodiments, the progranulin level to have a therapeutic effect is from 2 ng/mL to 4.5 ng/mL. In some embodiments, the progranulin level to have a therapeutic effect is from 1 ng/mL to 10 ng/mL. In some embodiments, a progranulin level in a serum of the subject is not more than 300 ng/mL.

[0214] In some embodiments, a recombinant polynucleotide of the present disclosure encoding GRN can be used to treat dementia. Treatment of dementia can comprise expressing progranulin in a subject. In some embodiments, treatment of dementia comprises expressing progranulin in a cerebrospinal fluid of a subject. The progranulin can be expressed in the cerebrospinal fluid at a level high enough to have a therapeutic effect. In some embodiments, the progranulin level high enough to have a therapeutic effect is at least 3 ng/mL. In some embodiments, the progranulin level to have a therapeutic effect is from 1 ng/mL to 3 ng/mL. In some embodiments, the progranulin level to have a therapeutic effect is from 2 ng/mL to 4.5 ng/mL. In some embodiments, the progranulin level to have a therapeutic effect is from 1 ng/mL to 10 ng/mL. In some embodiments, the progranulin level to have a therapeutic effect is from 1 ng/mL to 100 ng/mL. In some embodiments, the progranulin level to have a therapeutic effect is from 10 ng/mL to 100 ng/mL. In some embodiments, the progranulin level to have a therapeutic effect is from 1 ng/mL to 30 ng/mL. In some embodiments, the progranulin level to have a therapeutic effect is from 10 ng/mL to 50 ng/mL. In some embodiments, the progranulin level to have a therapeutic effect is from 10 ng/mL to 30 ng/mL. In some embodiments, the progranulin level to have a therapeutic effect is from 70 ng/mL to 180 ng/mL. In some embodiments, the progranulin level to have a therapeutic effect is from 70 ng/mL to 300 ng/mL. In some embodiments, the progranulin level to have a therapeutic effect is comparable to a progranulin level of a healthy patient. In some embodiments, a progranulin level in a serum of the subject is not more than 300 ng/mL.

Methods of Treatment

[0215] A recombinant polynucleotide, plasmid, vector, and/or pharmaceutical composition of the present disclosure can be used in a method of treating a disorder in a subject in need thereof. A disorder can be a disease, a condition, a genotype, a phenotype, or any state associated with an adverse effect. In some embodiments, treating a disorder can comprise preventing, slowing progression of, reversing, or alleviating symptoms of the disorder. A method of treating a disorder can comprise delivering a recombinant polynucleotide as disclosed herein to a cell of a subject in need thereof and expressing the payload in the cell. A method of treating a disorder can comprise delivering a recombinant polynucleotide as disclosed herein to a cell of a subject in need thereof and expressing the progranulin in the cell. In some embodiments, a recombinant polynucleotide of the present disclosure can be used to treat a genetic disorder (e.g., FTD). In some embodiments, a recombinant polynucleotide of the present disclosure can be used to treat a neurodegenerative disorder (e.g., FTD, ALS, Alzheimer’s Disease, Parkinson’s Disease, or dementia). In some embodiments, a recombinant polynucleotide of the present disclosure can be used to treat a condition associated with one or more mutations.

[0216] In some embodiments, the recombinant polynucleotides of the present disclosure express the payload, which results in increased protein expression levels corresponding to the payload in the cell, tissue, or subject. For example, a recombinant polynucleotide encoding progranulin can increase progranulin expression levels in a cell, tissue, or subject. The recombinant polynucleotides of the present disclosure produce a from 1.1 -fold to 1000-fold increased protein expression in the cell, tissue, or subject. The recombinant polynucleotides of the present disclosure produce a from 1.1 -fold to 1000-fold, from 1.5-fold to 1000-fold, from 2-fold to 1000-fold, from 5-fold to 1000-fold, from 10-fold to 1000-fold, from 20-fold to 1000-fold, from 50-fold to 1000-fold, from 100-fold to 1000-fold, from 200-fold to 1000-fold, from 500-fold to 1000-fold, from 1.1 -fold to 10-fold, from 1.5-fold to 10-fold, from 2-fold to 10-fold, from 5-fold to 10-fold, from 10-fold to 100-fold, from 20-fold to 100-fold, or from 50-fold to 100-fold increased protein expression in the cell, tissue, or subject. In some embodiments, the recombinant polynucleotides of the present disclosure produce at least 1.1 -fold, at least 1.5-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, or at least 500-fold increased protein expression in the cell, tissue, or subject. Increase in protein expression can be measured by an assay comparing a sample or subject treated with the recombinant polynucleotide to a control sample or subject not treated with the recombinant polynucleotide.

[0217] In some embodiments, a protein encoded by a recombinant polynucleotide is expressed at different levels in a first cell type, tissue type, or organ than in a second cell type, tissue type, or organ. For example, progranulin encoded by a recombinant polynucleotide is expressed at a different level in a cerebrospinal fluid of a subject than in a serum of a subject. In some embodiments, a recombinant polynucleotide expresses a protein in a first cell type, tissue type, or organ at a level that is at least 0.001 -fold, at least 0.005-fold, at least 0.01 -fold, at least 0.02- fold, at least 0.05-fold, at least 0.1 -fold, at least 0.2-fold, at least 0.25-fold, at least 0.5-fold, at least 1-fold, at least 2-fold, at least 5 -fold, at least 10-fold, at least 15 -fold, at least 20-fold, at least 25 -fold, at least 50-fold, at least 75 -fold, at least 100-fold, at least 200-fold, at least 500- fold, or at least 1000-fold an expression level of the protein in a second cell type, tissue type, or organ. [0218] In some embodiments, a protein encoded by a recombinant polynucleotide is expressed at different levels in a first cell type, tissue type, or organ than in a second cell type, tissue type, or organ. For example, progranulin encoded by a recombinant polynucleotide is expressed at a different level in a cerebrospinal fluid of a subject than in a serum of a subject. In some embodiments, a recombinant polynucleotide expresses progranulin in a first cell type, tissue type, or organ at a level that is at least 0.001 -fold, at least 0.005-fold, at least 0.01 -fold, at least 0.02-fold, at least 0.05-fold, at least 0.1 -fold, at least 0.2-fold, at least 0.25-fold, at least 0.5-fold, at least 1-fold, at least 2-fold, at least 5 -fold, at least 10-fold, at least 15 -fold, at least 20-fold, at least 25 -fold, at least 50-fold, at least 75 -fold, at least 100-fold, at least 200-fold, at least 500- fold, or at least 1000-fold higher than an expression level of the protein in a second cell type, tissue type, or organ. In some embodiments, a recombinant polynucleotide expresses progranulin in cerebral spinal fluid (CSF) at a level that is at least 0.001 -fold, at least 0.005-fold, at least 0.01 -fold, at least 0.02-fold, at least 0.05-fold, at least 0.1 -fold, at least 0.2-fold, at least 0.25-fold, at least 0.5-fold, at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 15 -fold, at least 20-fold, at least 25 -fold, at least 50-fold, at least 75 -fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold higher than an expression level of progranulin in serum. In some embodiments, a recombinant polynucleotide expresses progranulin in serum at a level that is at least 0.001 -fold, at least 0.005-fold, at least 0.01 -fold, at least 0.02-fold, at least 0.05-fold, at least 0.1 -fold, at least 0.2-fold, at least 0.25-fold, at least 0.5 -fold, at least 1-fold, at least 2-fold, at least 5 -fold, at least 10-fold, at least 15 -fold, at least 20-fold, at least 25 -fold, at least 50-fold, at least 75 -fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold higher than an expression level of progranulin in CSF. In some embodiments, a recombinant polynucleotide expresses progranulin in serum at a level that is from 20-fold to 500-fold higher than an expression level of progranulin in CSF. In some embodiments, a recombinant polynucleotide expresses progranulin in serum at a level that is from 20-fold to 200-fold higher than an expression level of progranulin in CSF. In some embodiments, a recombinant polynucleotide expresses progranulin in serum at a level that is from 20-fold to 100-fold higher than an expression level of progranulin in CSF. In some embodiments, a recombinant polynucleotide expresses progranulin in serum at a level that is from 20-fold to 50-fold higher than an expression level of progranulin in CSF. In some embodiments, a recombinant polynucleotide expresses progranulin in serum at a level that is from 20-fold to 30-fold higher than an expression level of progranulin in CSF. In some embodiments, a recombinant polynucleotide expresses progranulin in serum at a level that is from 1-fold to 200-fold higher than an expression level of progranulin in CSF. In some embodiments, a recombinant polynucleotide expresses progranulin in serum at a level that is from 1-fold to 50-fold higher than an expression level of progranulin in CSF. In some embodiments, a recombinant polynucleotide expresses progranulin in serum at a level that is from 1-fold to 25-fold higher than an expression level of progranulin in CSF. In some embodiments, a recombinant polynucleotide expresses progranulin in serum at a level that is from 1-fold to 20-fold higher than an expression level of progranulin in CSF. In some embodiments, a recombinant polynucleotide expresses progranulin in serum at a level that is from 1-fold to 15-fold higher than an expression level of progranulin in CSF. In some embodiments, a recombinant polynucleotide expresses progranulin in serum at a level that is from 1-fold to 10-fold higher than an expression level of progranulin in CSF. In some embodiments, a recombinant polynucleotide expresses progranulin in serum at a level that is from 1-fold to 5-fold higher than an expression level of progranulin in CSF. In some embodiments, a recombinant polynucleotide expresses progranulin in serum at a level that is from 1-fold to 3-fold higher than an expression level of progranulin in CSF. In some embodiments, a recombinant polynucleotide expresses progranulin in serum at a level that is from 1-fold to 2-fold higher than an expression level of progranulin in CSF. In some embodiments, a recombinant polynucleotide expresses progranulin in serum at a level that is from 0.001 -fold to 1-fold higher than an expression level of progranulin in CSF.

[0219] In some embodiments, a recombinant polynucleotide expresses a protein in a first cell type, tissue type, or organ at a level that is not less than 0.001 -fold and not more than 100-fold, not less than 0.01 -fold and not more than 100-fold, not less than 0.05-fold and not more than 100-fold, not less than 0.1 -fold and not more than 100-fold, not less than 0.2-fold and not more than 100-fold, not less than 0.25-fold and not more than 100-fold, not less than 0.5-fold and not more than 100-fold, not less than 1-fold and not more than 100-fold, not less than 5-fold and not less than 100-fold, not less than 10-fold and not more than 100-fold, not less than 0.001 -fold and not more than 50-fold, not less than 0.01 -fold and not more than 50-fold, not less than 0.05-fold and not more than 50-fold, not less than 0.1 -fold and not more than 50-fold, not less than 0.2- fold and not more than 50-fold, not less than 0.25 -fold and not more than 50-fold, not less than 0.5 -fold and not more than 50-fold, not less than 1-fold and not more than 50-fold, not less than 5 -fold and not more than 50-fold, not less than 10-fold and not more than 50-fold, not less than 0.001 -fold and not more than 10-fold, not less than 0.01 -fold and not more than 10-fold, not less than 0.05-fold and not more than 10-fold, not less than 0.1 -fold and not more than 10-fold, not less than 0.2-fold and not more than 10-fold, not less than 0.25-fold and not more than 10-fold, not less than 0.5-fold and not more than 10-fold, not less than 1-fold and not more than 10-fold, or not less than 5-fold and not more than 10-fold an expression level of the protein in a second cell type, tissue type, or organ.

[0220] In some embodiments, a recombinant polynucleotide expresses progranulin in a first cell type, tissue type, or organ at a level that is not less than 0.001 -fold and not more than 100-fold, not less than 0.01 -fold and not more than 100-fold, not less than 0.05-fold and not more than 100-fold, not less than 0.1 -fold and not more than 100-fold, not less than 0.2-fold and not more than 100-fold, not less than 0.25-fold and not more than 100-fold, not less than 0.5-fold and not more than 100-fold, not less than 1-fold and not more than 100-fold, not less than 5-fold and not more than 100-fold, not less than 10-fold and not more than 100-fold, not less than 0.001 -fold and not more than 50-fold, not less than 0.01 -fold and not more than 50-fold, not less than 0.05- fold and not more than 50-fold, not less than 0.1-fold and not more than 50-fold, not less than 0.2-fold and not more than 50-fold, not less than 0.25-fold and not more than 50-fold, not less than 0.5 -fold and not more than 50-fold, not less than 1-fold and not more than 50-fold, not less than 5-fold and not more than 50-fold, not less than 10-fold and not more than 50-fold, not less than 0.001 -fold and not more than 10-fold, not less than 0.01 -fold and not more than 10-fold, not less than 0.05-fold and not more than 10-fold, not less than 0.1-fold and not more than 10-fold, not less than 0.2-fold and not more than 10-fold, not less than 0.25 -fold and not more than 10- fold, not less than 0.5-fold and not more than 10-fold, not less than 1-fold and not more than 10- fold, or not less than 5-fold and not more than 10-fold higher than an expression level of progranulin in a second cell type, tissue type, or organ. A first cell type can be a blood cell. A first tissue type can be serum. A first organ can be a spleen or liver. A second cell type can be a neuronal cell type. A second tissue type can be a neuronal tissue. A second organ can be a central nervous system organ, e.g., brain or spinal cord.

Treatment of Diseases associated with GRN

[0221] An exemplary gene associated with a disease or condition that can be treated by a recombinant polynucleotide as described herein is GRN. An exemplary disease or condition associated with GRN is frontotemporal dementia (FTD). Additional diseases or conditions associated with GRN that can be treated using a recombinant polynucleotide of the present disclosure include amyotrophic lateral sclerosis (ALS), Alzheimer’s Disease, Parkinson’s Disease, stroke, Gaucher disease, arthritis, limbic-predominant age-related transactivation response DNA-binding protein 43 (TDP-43) encephalopathy, autism, neuronal ceroid lipofuscinosis (e.g., type 11 (CLN11)), dementia, and neurodegeneration, such as neurodegeneration associated with normal brain aging. [0222] Progranulin, encoded by GRN, is a precursor protein cleaved to form granulin. GRN is expressed in peripheral and central nervous system tissues and is upregulated in microglia following injury. Both granulin and progranulin are implicated in a wide variety of functions, including development, inflammation, cell proliferation, and protein homeostasis. Mutations in GRN are implicated in frontotemporal dementia, amyotrophic lateral sclerosis (ALS), Alzheimer’s Disease, Parkinson’s Disease, stroke, Gaucher disease, arthritis, limbic- predominant age-related transactivation response DNA-binding protein 43 (TDP-43) encephalopathy, autism, neuronal ceroid lipofuscinosis (e.g., type 11 (CLN11)), dementia, and neurodegeneration. Additionally, GRN has been shown to be anti-inflammatory. Therefore, GRN can potentially aid disease treatment as anti-inflammatory, such as for treating frontotemporal dementia, amyotrophic lateral sclerosis (ALS), Alzheimer’s Disease, Parkinson’s Disease, stroke, Gaucher disease, arthritis, limbic-predominant age-related transactivation response DNA-binding protein 43 (TDP-43) encephalopathy, autism, neuronal ceroid lipofuscinosis (e.g., type 11 (CLN11)), dementia, and neurodegeneration. Furthermore, GRN can potentially aid disease treatment as anti-inflammatory for patients with normal GRN, such as for treating neurodegeneration caused by normal aging. Described herein are methods of increasing expression of GRN using recombinant polynucleotide expressing progranulin to treat a disease (e.g., amyotrophic lateral sclerosis (ALS), Alzheimer’s Disease, Parkinson’s Disease, stroke, Gaucher disease, arthritis, limbic-predominant age-related transactivation response DNA-binding protein 43 (TDP-43) encephalopathy, autism, neuronal ceroid lipofuscinosis (e.g., type 11 (CLN11)), dementia, and neurodegeneration, such as neurodegeneration associated with normal brain aging).

[0223] Mutations in GRN can lead to frontotemporal dementia (FTD), a neurodegenerative disease. Loss of function mutations leading to GRN haploinsufficiency can be implicated in familial and sporadic cases of frontotemporal dementia. GRN has anti-inflammatory properties, so expression of GRN can reduce inflammation, protecting against neurodegeneration. In FTD cases, decreased levels of progranulin are observed in the serum and cerebrospinal fluid of subjects with loss of function mutations and subjects without said mutations. Thus, the recombinant polynucleotides of the present disclosure can increase expression of progranulin as a means to restore or enhance progranulin levels. ALS, Alzheimer’s Disease, Parkinson’s Disease, and dementia also associated with loss of function GRN mutations. Thus, increasing expression of GRN can be used to treat LS, Alzheimer’s Disease, Parkinson’s Disease, and dementia. [0224] In some embodiments, the recombinant polynucleotides of the present disclosure express progranulin as the payload, which results in increased progranulin expression levels in the cell, tissue, or subject. The recombinant polynucleotides of the present disclosure produce a from 1.1- fold to 1000-fold increased progranulin expression in the cell, tissue, or subject. The recombinant polynucleotides of the present disclosure produce a from 1.1 -fold to 1000-fold, from 1.5-fold to 1000-fold, from 2-fold to 1000-fold, from 5-fold to 1000-fold, from 10-fold to 1000-fold, from 20-fold to 1000-fold, from 50-fold to 1000-fold, from 100-fold to 1000-fold, from 200-fold to 1000-fold, from 500-fold to 1000-fold, from 1.1 -fold to 10-fold, from 1.5-fold to 10-fold, from 2-fold to 10-fold, from 5-fold to 10-fold, from 10-fold to 100-fold, from 20-fold to 100-fold, or from 50-fold to 100-fold increased progranulin expression in the cell, tissue, or subject. In some embodiments, the recombinant polynucleotides of the present disclosure produce at least 1.1 -fold, at least 1.5-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, or at least 500-fold increased progranulin expression in the cell, tissue, or subject. Increase in progranulin expression can be measured by an assay comparing a sample or subject treated with the recombinant polynucleotide to a control sample or subject not treated with the recombinant polynucleotide. [0225] In some embodiments, a recombinant polynucleotide expresses progranulin in cerebrospinal fluid at a level that is at least 0.001 -fold, at least 0.005-fold, at least 0.01 -fold, at least 0.02-fold, at least 0.05-fold, at least 0.1 -fold, at least 0.2-fold, at least 0.25-fold, at least 0.5 -fold, at least 1-fold, at least 2-fold, at least 5 -fold, at least 10-fold, at least 15 -fold, at least 20-fold, at least 25 -fold, at least 50-fold, at least 75 -fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold an expression level of progranulin in serum.

[0226] In some embodiments, a recombinant polynucleotide expresses progranulin in serum at a level that is no more than 0.001 -fold, no more than 0.005-fold, no more than 0.01 -fold, no more than 0.02-fold, no more than 0.05-fold, no more than 0.1-fold, no more than 0.2-fold, no more than 0.25-fold, no more than 0.5-fold, no more than 1-fold, no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25- fold, no more than 50-fold, no more than 75 -fold, no more than 100-fold, no more than 200-fold, no more than 500-fold, or no more than 1000-fold an expression level of progranulin in cerebrospinal fluid.

[0227] In some embodiments, a recombinant polynucleotide expresses progranulin in cerebrospinal fluid at a level that is not less than 0.001 -fold and not more than 100-fold, not less than 0.01 -fold and not more than 100-fold, not less than 0.05-fold and not more than 100-fold, not less than 0.1 -fold and not more than 100-fold, not less than 0.2-fold and not more than 100- fold, not less than 0.25-fold and not more than 100-fold, not less than 0.5-fold and not more than 100-fold, not less than 1-fold and not more than 100-fold, not less than 5-fold and not more than 100-fold, not less than 10-fold and not more than 100-fold, not less than 0.001 -fold and not more than 50-fold, not less than 0.01 -fold and not more than 50-fold, not less than 0.05-fold and not more than 50-fold, not less than 0.1 -fold and not more than 50-fold, not less than 0.2-fold and not more than 50-fold, not less than 0.25-fold and not more than 50-fold, not less than 0.5-fold and not more than 50-fold, not less than 1-fold and not more than 50-fold, not less than 5-fold and not more than 50-fold, not less than 10-fold and not more than 50-fold, not less than 0.001- fold and not more than 10-fold, not less than 0.01 -fold and not more than 10-fold, not less than 0.05 -fold and not more than 10-fold, not less than 0.1 -fold and not more than 10-fold, not less than 0.2-fold and not more than 10-fold, not less than 0.25-fold and not more than 10-fold, not less than 0.5-fold and not more than 10-fold, not less than 1-fold and not more than 10-fold, or not less than 5-fold and not more than 10-fold an expression level of progranulin in serum. [0228] In some embodiments, a recombinant polynucleotide expresses progranulin in serum at a level that is not less than 0.001 -fold and not more than 100-fold, not less than 0.01 -fold and not more than 100-fold, not less than 0.05-fold and not more than 100-fold, not less than 0.1 -fold and not more than 100-fold, not less than 0.2-fold and not more than 100-fold, not less than 0.25-fold and not more than 100-fold, not less than 0.5-fold and not more than 100-fold, not less than 1-fold and not more than 100-fold, not less than 5-fold and not more than 100-fold, not less than 10-fold and not more than 100-fold, not less than 0.001 -fold and not more than 50-fold, not less than 0.01 -fold and not more than 50-fold, not less than 0.05 -fold and not more than 50-fold, not less than 0.1 -fold and not more than 50-fold, not less than 0.2-fold and not more than 50- fold, not less than 0.25-fold and not more than 50-fold, not less than 0.5-fold and not more than 50-fold, not less than 1-fold and not more than 50-fold, not less than 5-fold and not more than 50-fold, not less than 10-fold and not more than 50-fold, not less than 0.001 -fold and not more than 10-fold, not less than 0.01 -fold and not more than 10-fold, not less than 0.05-fold and not more than 10-fold, not less than 0.1 -fold and not more than 10-fold, not less than 0.2-fold and not more than 10-fold, not less than 0.25-fold and not more than 10-fold, not less than 0.5-fold and not more than 10-fold, not less than 1-fold and not more than 10-fold, or not less than 5-fold and not more than 10-fold higher than an expression level of progranulin in the cerebrospinal fluid.

Recombinant Vectors and Delivery

[0229] In some embodiments, a recombinant polynucleotide of the present disclosure is introduced into a subject via a delivery vehicle. In some embodiments, the delivery vehicle is a vector. In some embodiments the vector is a plasmid, a viral vector, or a transformed cell. A vector can facilitate delivery of the recombinant polynucleotide into a cell for expressing the payload (e.g., progranulin). In some examples, the vector comprises DNA, such as double stranded or single stranded DNA. In some examples, the delivery vector can be a eukaryotic vector, a prokaryotic vector (e.g., a bacterial vector or plasmid), a viral vector, or any combination thereof. In some embodiments, a viral vector comprises a viral capsid, an inverted terminal repeat sequence, and the engineered polynucleotide can be used to deliver the recombinant polynucleotide to a cell.

[0230] In some embodiments, the viral vector can be a retroviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, an alphavirus vector, a lentivirus vector (e.g., human or porcine), a Herpes virus vector, an Epstein-Barr virus vector, an SV40 virus vectors, a pox virus vector, or a combination thereof. In some embodiments, the viral vector can be a recombinant vector, a hybrid vector, a chimeric vector, a self-complementary vector, a single-stranded vector, or any combination thereof.

[0231] In some embodiments, the viral vector is an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector. Adeno-associated virus (AAV) vectors include vectors derived from any AAV serotype, including, but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-DJ/8, AAV-DJ/9, AAV1/2, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAV.OligoOOl, AAV.SCH9, AAV.r3.45, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PhP.eB, AAV.PhP.Vl, AAV.PHP.B, AAV.PhB.Cl, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, and AAVhu68.

[0232] In some embodiments, a polynucleotide is introduced into a subject by non-viral vector systems. In some embodiments, cationic lipids, polymers, hydrodynamic injection and/or ultrasound can be used in delivering a polynucleotide to a subject in the absence of virus.

[0233] In some examples, the vector can be a eukaryotic vector, a prokaryotic vector (e.g., a bacterial vector) a viral vector, or any combination thereof. In some examples, the vector can be a viral vector. In some embodiments, the viral vector can be a retroviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, an alphavirus vector, a lentivirus vector (e.g., human or porcine), a Herpes virus vector, an Epstein-Barr virus vector, an SV40 virus vectors, a pox virus vector, or a combination thereof. In some embodiments, the viral vector can be a recombinant vector, a hybrid vector, a chimeric vector, a self-complementary vector, a singlestranded vector, or any combination thereof.

[0234] In some embodiments, the viral vector can be an adeno-associated virus (AAV). In some embodiments, the AAV can be any AAV known in the art. In some embodiments, the viral vector can be of a specific serotype. In some embodiments, the viral vector can be an AAV 1 serotype, AAV2 serotype, AAV3 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, AAV 10 serotype, AAV 11 serotype, AAV 12 serotype, AAV13 serotype, AAV14 serotype, AAV15 serotype, AAV16 serotype, AAV-DJ serotype, AAV-DJ/8 serotype, AAV-DJ/9 serotype, AAV1/2 serotype, AAV.rh8 serotype, AAV.rhlO serotype, AAV.rh20 serotype, AAV.rh39 serotype, AAV.Rh43 serotype, AAV.Rh74 serotype, AAV.v66 serotype, AAV.OligoOOl serotype, AAV.SCH9 serotype, AAV.r3.45 serotype, AAV.RHM4-1 serotype, AAV.hu37 serotype, AAV.Anc80 serotype, AAV.Anc80L65 serotype, AAV.7m8 serotype, AAV.PhP.eB serotype, AAV.PhP.Vl serotype, AAV.PHP.B serotype, AAV.PhB.Cl serotype, AAV.PhB.C2 serotype, AAV.PhB.C3 serotype, AAV.PhB.C6 serotype, AAV.cy5 serotype, AAV2.5 serotype, AAV2tYF serotype, AAV3B serotype, AAV.LK03 serotype, AAV.HSC1 serotype, AAV.HSC2 serotype, AAV.HSC3 serotype, AAV.HSC4 serotype, AAV.HSC5 serotype, AAV.HSC6 serotype, AAV.HSC7 serotype, AAV.HSC8 serotype, AAV.HSC9 serotype, AAV.HSC10 serotype, AAV.HSC11 serotype, AAV.HSC12 serotype, AAV.HSC13 serotype, AAV.HSC14 serotype, AAV.HSC15 serotype, AAV.HSC16 serotype, AAV.HSC17 serotype, or AAVhu68 serotype, a derivative of any of these serotypes, or any combination thereof.

[0235] In some embodiments, the AAV vector can be a recombinant vector, a hybrid AAV vector, a chimeric AAV vector, a self-complementary AAV (scAAV) vector, a single-stranded AAV (ssAAV), or any combination thereof.

[0236] In some embodiments, the AAV vector can be a recombinant AAV (rAAV) vector or engineered AAV vector. Methods of producing recombinant AAV vectors can be known in the art and generally involve, in some cases, introducing into a producer cell line: (1) DNA necessary for AAV replication and synthesis of an AAV capsid, (b) one or more helper constructs comprising the viral functions missing from the AAV vector, (c) a helper virus, and (d) the plasmid construct containing the genome of the AAV vector, e.g., ITRs, promoter and payload sequences, etc. (e.g., the plasmid of SEQ ID NO: 9). In some examples, the viral vectors described herein can be engineered through synthetic or other suitable means by references to published sequences, such as those that may be available in the literature. For example, the genomic and protein sequences of various serotypes of AAV, as well as the sequences of the native terminal repeats (TRs), Rep proteins, and capsid subunits may be known in the art and can be found in the literature or in public databases such as GenBank or Protein Data Bank (PDB).

[0237] An AAV vector can be assembled from one or more viral capsid proteins (VP). Viral capsid protein is referred to as AAV5 VP1 when referencing AAV5 VP1 positional notation. In all cases, viral capsid sequences and mutations disclosed herein should be understood as pertaining to all isoforms of the capsid protein (VP1, VP2, and VP3), as a mixture of these isoforms assemble to form virions. The positional amino acid residue designations “581 to 589” are relative to the translational start of the AAV5 VP1 polypeptide and should be adjusted accordingly to the relative start sites of AAV5 VP2 and AAV5 VP3. It should be understood that the present disclosure, when describing any particular VP1 sequence with mutations at particular amino acid residue positions, necessarily also encompasses corresponding mutations in VP2 and VP3. For example, any consensus sequence or specific sequence of an AAV5 VP1 capsid protein having one or more mutations in the 581-589 region, corresponding to amino acid residues 581 to 589 of AAV5 VP1, also encompasses AAV5 VP2 and AAV5 VP3 capsid proteins having said one or more mutations in an amino acid residue region in VP2 and VP3 corresponding to the amino acid residues of the VP1 581 to 589 region. For example, the amino acid residues of the 581 to 589 region of AAV5 VP1 (SEQ ID NO: 75;

MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNG LDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLG KAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPS GSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRV VTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRD WQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVV GNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFT YNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKN WFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSN TYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSST TAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPM MLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYN DPQFVDFAPDSTGEYRTTRPIGTRYLTRPL) correspond to the amino acid residues of the 445 to 453 region of AAV5 VP2 (SEQ ID NO: 76;

TAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAG GGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKS GSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQ VKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGY ATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLAN PLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRA SVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATY LEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERD VYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQ YSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRY LTRPL) and to the amino acid residues of 389 to 397 region of AAV5 VP3 (SEQ ID NO: 77;

MSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQY REIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVK IFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQ YGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFK LANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGV NRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTT ATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWM ERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSS FITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIG TRYLTRPL). As used herein, “wild type AAV5” or “wild type AAV5 capsid polypeptide” refers to an AAV5 VP1 capsid polypeptide of SEQ ID NO: 75, an AAV5 VP2 capsid polypeptide of SEQ ID NO: 76, an AAV5 VP3 capsid polypeptide of SEQ ID NO: 77, or a combination thereof. Also as used herein, a “wild type 581-589 region” refers to a 581 to 589 region of AAV5 VP1 having a sequence of ATGTYNLQE (SEQ ID NO: 79).

[0238] As used herein, “581-589 region” refers to a region or fragment of AAV5 VP1 corresponding to amino acid residues 581 to 589 relative to the translational start of the AAV5 VP1 polypeptide. A 581-589 region comprising at least one mutation relative to a wild type 581- 589 region sequence (e.g., a 581-589 region of a wild type AAV5 VP1 capsid polypeptide) can also be referred to as a “variant region” or a “variant 581-589 region.” The 581-589 region corresponds to amino acid residues 445 to 453 of AAV5 VP2 and to amino acid residues 389 to 397 of AAV5 VP3. The 581-589 region can confer tissue tropism to an AAV, and defined variants can be engineered to confer tissue tropism to an rAAV formed from viral capsid polypeptides (VP1, VP2, and VP3) comprising the 581-589 region. The AAV5 VP1 with a generalized 581-589 region is provided in SEQ ID NO: 78 (MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNG LDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLG KAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPS GSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRV VTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRD WQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVV GNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFT YNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKN WFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSN TYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSST TAPX¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹IVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLK HPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYT NNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL), in which the 581-589 region has a sequence of X¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹; wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, and X⁹ are each independently selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V. [0239] In some embodiments, an engineered adeno-associated virus (AAV) viral protein (VP) capsid polypeptide has an amino acid sequence at least 70% identical to an AAV5 VP capsid polypeptide (e.g., a VP capsid polypeptide of SEQ ID NO: 75, SEQ ID NO: 76, or SEQ ID NO: 77), wherein the engineered AAV5 VP capsid polypeptide has at least one substitution as compared to SEQ ID NO: 75 in the 581-589 region, corresponding to residue 581 to residue 589 of SEQ ID NO: 75, inclusive, wherein the capsid polypeptide is capable of assembling into a recombinant AAV5 virion (rAAV5). In some embodiments, the AAV5 VP capsid polypeptide comprises a variant 581-589 region (e.g., comprising one or more amino acid substitutions relative to SEQ ID NO: 75 in the region from residue 581 to residue 589, inclusive). The AAV5 VP capsid polypeptide can comprise a sequence of SEQ ID NO: 78, wherein the 581-589 region has a one or more mutations relative to the wild type AAV5 VP1 capsid polypeptide, wherein the one or more mutations confers tissue tropism (e.g., CNS tissue tropism). For example, an AAV5 VP capsid polypeptide can comprise a sequence of SEQ ID NO: 78, wherein the 581-589 region has a sequence of any one of SEQ ID NO: 82 - SEQ ID NO: 111. In some embodiments, the 581-589 region confers tropism for a tissue of interest (e.g., CNS tissue). Examples of 581- 589 region sequences that can be included in an engineered VP capsid polypeptide (e.g., an engineered AAV5 VP capsid polypeptide) are provided in TABLE 6. In some embodiments, a 581-589 region sequence provided in TABLE 6 can confer CNS tissue tropism on a rAAV assembled from engineered VP capsid polypeptides including the 581-589 region sequence. TABLE 6. Exemplary 581-589 Region Sequences

[0240] In particular embodiments, the engineered adeno-associated virus (AAV) viral protein (VP) capsid polypeptide has an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identical to an AAV5 VP capsid polypeptide (e.g., a VP capsid polypeptide of SEQ ID NO: 75, SEQ ID NO: 76, or SEQ ID NO: 77).

[0241] In some embodiments, the AAV VP capsid polypeptides have an amino acid sequence of SEQ ID NO: 78, wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, and X⁹ are each independently selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V. In some embodiments, the AAV VP capsid polypeptide has an amino acid sequence of SEQ ID NO: 78, wherein the X¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹ portion corresponds to a sequence selected from any one of SEQ ID NO: 82 - SEQ ID NO: 111.

[0242] In some embodiments, the 581-589 region of the engineered VP capsid polypeptide, corresponding to residues 581 to 589, inclusive, with reference to SEQ ID NO: 75, has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical to any one of SEQ ID NO: 82 - SEQ ID NO: 111, preferably to any one of SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 96, or SEQ ID NO: 82. In some embodiments, the 581-589 region of the engineered VP capsid polypeptide comprises 1 amino acid substitution relative to any one of SEQ ID NO: 82 - SEQ ID NO: 111, preferably to any one of SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 96, or SEQ ID NO: 82. In some embodiments, the 581-589 region of the engineered VP capsid polypeptide comprises 2 amino acid substitutions relative to any one of SEQ ID NO: 82 - SEQ ID NO: 111, preferably to any one SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 96, or SEQ ID NO: 82. In particular embodiments, the 581-589 region of the engineered VP capsid polypeptide has a sequence that is identical to any one of SEQ ID NO: 82 - SEQ ID NO: 111, preferably to any one of SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 96, or SEQ ID NO: 82.

[0243] In some embodiments, the engineered AAV VP capsid polypeptide comprises a polypeptide sequence represented by the formula: (A)-(X)-(B) wherein: (A) is the polypeptide sequence of SEQ ID NO: 80 (MSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQ YREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRV KIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLP QYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLF KLANPL VDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSG VNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGT TATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAP, corresponding to residues 193 to 580 of VP1 (SEQ ID NO: 75), residues 57 to 444 of VP2 (SEQ ID NO: 76), or residues 1 to 388 of VP3 (SEQ ID NO: 77)); (X) is the 581-589 region having a polypeptide sequence of any one of SEQ ID NO: 82 - SEQ ID NO: 111; and (B) is the polypeptide sequence of SEQ ID NO: 81 (IVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNI TSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDST GEYRTTRPIGTRYLTRPL, corresponding to residues 590 to 724 of VP1 (SEQ ID NO: 75), residues 454 to 588 of VP2 (SEQ ID NO: 76), or residues 398 to 532 of VP3 (SEQ ID NO: 77)). [0244] In some embodiments, the engineered AAV VP capsid polypeptide comprises a polypeptide sequence represented by the formula: (A)-(X)-(B) wherein: (A) is the polypeptide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 80; (X) is the 581-589 region having a polypeptide sequence selected from the list of polypeptides of any one of SEQ ID NO: 82 - SEQ ID NO: 111, preferably any one of SEQ ID NO: 83 or SEQ ID NO 84; and (B) is the polypeptide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 81.

[0245] In some examples, methods of producing delivery vectors herein comprising packaging a polynucleotide of the present disclosure in an AAV vector (e.g., a recombinant AAV5 comprising an engineered AAV5 VP capsid polypeptide). In some examples, methods of producing the delivery vectors described herein comprise, (a) introducing into a cell: (i) a polynucleotide disclosed herein (e.g., a plasmid of SEQ ID NO: 9); and (ii) a viral genome comprising a Replication (Rep) gene and Capsid (Cap) gene that encodes a wild type AAV capsid protein or modified version thereof; (b) expressing in the cell the wild type AAV capsid protein or modified version thereof; (c) assembling an AAV particle; and (d) packaging the recombinant polynucleotide disclosed herein in the AAV particle, thereby generating an AAV delivery vector. In some examples, any polynucleotide disclosed herein can be packaged in the AAV vector. In some examples, the recombinant vectors comprise one or more inverted terminal repeats and the inverted terminal repeats comprise a 5 ’ inverted terminal repeat, a 3 ’ inverted terminal repeat, and a mutated inverted terminal repeat. In some examples, the mutated terminal repeat lacks a terminal resolution site, thereby enabling formation of a self- complementary AAV.

[0246] In some examples, a hybrid AAV vector can be produced by transcapsidation, e.g., packaging an inverted terminal repeat (ITR) from a first serotype into a capsid of a second serotype, wherein the first and second serotypes may be not the same. In some examples, the Rep gene and ITR from a first AAV serotype (e.g., AAV2) can be used in a capsid from a second AAV serotype (e.g., AAV5 or AAV9), wherein the first and second AAV serotypes may not be the same. As a non-limiting example, a hybrid AAV serotype comprising the AAV2 ITRs and AAV9 capsid protein may be indicated AAV2/9. In some examples, the hybrid AAV delivery vector comprises an AAV2/1, AAV2/2, AAV 2/4, AAV2/5, AAV2/8, or AAV2/9 vector.

[0247] In some examples, the AAV vector can be a chimeric AAV vector. In some examples, the chimeric AAV vector comprises an exogenous amino acid or an amino acid substitution, or capsid proteins from two or more serotypes. In some examples, a chimeric AAV vector can be genetically engineered to increase transduction efficiency, selectivity, or a combination thereof. [0248] In some examples, the AAV vector comprises a self-complementary AAV genome. Self- complementary AAV genomes may be generally known in the art and contain both DNA strands which can anneal together to form double-stranded DNA. TABLE 7 shows exemplary sequences of plasmids comprising a recombinant polynucleotide, which can be used to produce an encapsidated recombinant polynucleotide by the methods described herein (e.g., transient transfection to produce virus). The plasmid of SEQ ID NO: 9 encodes a recombinant polynucleotide comprising a CAG promoter operably linked to a coding sequence encoding human progranulin, further comprising a WPRE downstream of the coding sequence encoding human progranulin, wherein the recombinant polynucleotide is located within two flanking ITRs. The plasmid of SEQ ID NO: 19 encodes a recombinant polynucleotide comprising a RSV promoter operably linked to a coding sequence encoding human progranulin, further comprising a SV40 intron upstream of the coding sequence encoding human progranulin, wherein the recombinant polynucleotide is located within two flanking ITRs and the coding sequence encoding human progranulin comprises no introns (e.g., only comprises exons). The plasmid of SEQ ID NO: 20 encodes a recombinant polynucleotide comprising a RSV promoter operably linked to a coding sequence encoding human progranulin, further comprising a GRN intron 9 in the sequence human progranulin (e.g., all introns except GRN intron 9 were removed), wherein the recombinant polynucleotide is located within two flanking ITRs.

TABLE 7. Exemplary Plasmid Sequences

-Ill-

[0249] In a preferred embodiment, a plasmid for expressing progranulin comprises a sequence of SEQ ID NO: 120. SEQ ID NO: 120 comprises a recombinant polynucleotide of SEQ ID NO: 65. SEQ ID NO: 120 comprises a recombinant polynucleotide comprising a 5’ inverted terminal repeat (“5’ ITR”) of SEQ ID NO: 59, a 5’ stuffer sequence of SEQ ID NO: 61, an RSV promoter of SEQ ID NO: 3, a 5’ untranslated region of SEQ ID NO: 62 (which overlaps with the RSV promoter and comprises 33 nucleotides of the 3’ end of the RSV promoter and a Kozak sequence of SEQ ID NO: 66), a progranulin coding sequence of SEQ ID NO: 10 (comprising a sequence of SEQ ID NO: 57 encoding a progranulin signal peptide and a sequence of SEQ ID NO: 58), a Hindlll restriction site, a WPRE3 post-transcriptional regulatory element of SEQ ID NO: 8, a Xball restriction site, a polyadenylation signal of SEQ ID NO: 42, a Notl restriction site, a transcriptional pause site of SEQ ID NO: 64, a Sall restriction site, a PstI restriction site, and a 3’ inverted terminal repeat of SEQ ID NO: 60. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 80% sequence identity to SEQ ID NO: 120. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 85% sequence identity to SEQ ID NO: 120. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 90% sequence identity to SEQ ID NO: 120. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 95% sequence identity to SEQ ID NO: 120. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 96% sequence identity to SEQ ID NO: 120. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 97% sequence identity to SEQ ID NO: 120. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 98% sequence identity to SEQ ID NO: 120. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 99% sequence identity to SEQ ID NO: 120. In some embodiments, the plasmid comprising the recombinant polynucleotide comprises 100% sequence identity to SEQ ID NO: 120. In some embodiments, the plasmid comprising the recombinant polynucleotide is SEQ ID NO: 120. In some embodiments, the recombinant polynucleotide has at least 60%, 65%, 70% 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence comprising the recombinant polynucleotide between the ITRs of SEQ ID NO: 120.

[0250] Another example of a plasmid for expressing progranulin is SEQ ID NO: 9. A sequence map of SEQ ID NO: 9 is provided in FIG. 1. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 80% sequence identity to SEQ ID NO: 9. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 85% sequence identity to SEQ ID NO: 9. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 90% sequence identity to SEQ ID NO: 9. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 95% sequence identity to SEQ ID NO: 9. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 96% sequence identity to SEQ ID NO: 9. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 97% sequence identity to SEQ ID NO: 9. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 98% sequence identity to SEQ ID NO: 9. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 99% sequence identity to SEQ ID NO: 9. In some embodiments, the plasmid comprising the recombinant polynucleotide has comprise 100% sequence identity to SEQ ID NO: 9. In some embodiments, the plasmid comprising the recombinant polynucleotide is SEQ ID NO: 9. In some embodiments, the recombinant polynucleotide has at least 60%, 65%, 70% 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence comprising the recombinant polynucleotide between the ITRs of SEQ ID NO: 9.

[0251] Another example of plasmid for expressing progranulin is SEQ ID NO: 19. Nucleotides 402-498 of SEQ ID NO: 19 correspond to the left ITR (also referred to as the 5’ ITR). Nucleotides 505-766 of SEQ ID NO: 19 correspond to the RSV promoter. Nucleotides 774-872 of SEQ ID NO: 19 correspond to the SV40 intron. Nucleotides 888-2,669 of SEQ ID NO: 19 correspond to the GRN coding sequence. Nucleotides 2,670-2,725 of SEQ ID NO: 19 correspond to the rabbit beta globin polyA sequence. Nucleotides 2,876-2,732 of SEQ ID NO: 19 correspond to the right ITR (also referred to as the 3’ ITR). In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 80% sequence identity to SEQ ID NO: 19. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 85% sequence identity to SEQ ID NO: 19. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 90% sequence identity to SEQ ID NO: 19. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 95% sequence identity to SEQ ID NO: 19. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 96% sequence identity to SEQ ID NO: 19. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 97% sequence identity to SEQ ID NO: 19. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 98% sequence identity to SEQ ID NO: 19. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 99% sequence identity to SEQ ID NO: 19. In some embodiments, the plasmid comprising the recombinant polynucleotide comprises 100% sequence identity to SEQ ID NO: 19. In some embodiments, the plasmid comprising the recombinant polynucleotide is SEQ ID NO: 19. In some embodiments, the recombinant polynucleotide has at least 60%, 65%, 70% 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence comprising the recombinant polynucleotide between the ITRs of SEQ ID NO: 19.

[0252] Another example of plasmid for expressing progranulin is SEQ ID NO: 20. Nucleotides 402-498 of SEQ ID NO: 20 correspond to the left ITR. Nucleotides 505-766 of SEQ ID NO: 20 correspond to the RSV promoter. Nucleotides 782-2,652 of SEQ ID NO: 20 correspond to the GRN coding sequence. Nucleotides 1,715-1,803 of SEQ ID NO: 20 correspond to the GRN intron9. Nucleotides 2,653-2,708 of SEQ ID NO: 20 correspond to the RabBetaGlobinPolyA sequence. Nucleotides 2,859-2,715 of SEQ ID NO: 20 correspond to the right ITR. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 80% sequence identity to SEQ ID NO: 20. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 85% sequence identity to SEQ ID NO: 20. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 90% sequence identity to SEQ ID NO: 20. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 95% sequence identity to SEQ ID NO: 20. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 96% sequence identity to SEQ ID NO: 20. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 97% sequence identity to SEQ ID NO: 20. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 98% sequence identity to SEQ ID NO: 20. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 99% sequence identity to SEQ ID NO: 20. In some embodiments, the plasmid comprising the recombinant polynucleotide comprises 100% sequence identity to SEQ ID NO: 20. In some embodiments, the plasmid comprising the recombinant polynucleotide is SEQ ID NO: 20. In some embodiments, the recombinant polynucleotide has at least 60%, 65%, 70% 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence comprising the recombinant polynucleotide between the ITRs of SEQ ID NO: 20.

[0253] Another example of plasmid for expressing progranulin is SEQ ID NO: 49. The plasmid of SEQ ID NO: 49 contains inverted terminal repeats flanking a sequence encoding a JeT promoter, a sequence encoding progranulin, and a rabbit beta globin polyA signal. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 80% sequence identity to SEQ ID NO: 49. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 85% sequence identity to SEQ ID NO: 49. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 90% sequence identity to SEQ ID NO: 49. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 95% sequence identity to SEQ ID NO: 49. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 96% sequence identity to SEQ ID NO: 49. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 97% sequence identity to SEQ ID NO: 49. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 98% sequence identity to SEQ ID NO: 49. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 99% sequence identity to SEQ ID NO: 49. In some embodiments, the plasmid comprising the recombinant polynucleotide comprises 100% sequence identity to SEQ ID NO: 49. In some embodiments, the plasmid comprising the recombinant polynucleotide is SEQ ID NO: 49. In some embodiments, the recombinant polynucleotide has at least 60%, 65%, 70% 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence comprising the recombinant polynucleotide between the ITRs of SEQ ID NO: 49.

[0254] Another example of plasmid for expressing progranulin is SEQ ID NO: 50. The plasmid of SEQ ID NO: 50 contains inverted terminal repeats flanking a sequence encoding an RSV promoter, a sequence encoding progranulin, and a rabbit beta globin polyA signal. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 80% sequence identity to SEQ ID NO: 50. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 85% sequence identity to SEQ ID NO: 50. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 90% sequence identity to SEQ ID NO: 50. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 95% sequence identity to SEQ ID NO: 50. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 96% sequence identity to SEQ ID NO: 50. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 97% sequence identity to SEQ ID NO: 50. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 98% sequence identity to SEQ ID NO: 50. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 99% sequence identity to SEQ ID NO: 50. In some embodiments, the plasmid comprising the recombinant polynucleotide comprises 100% sequence identity to SEQ ID NO: 50. In some embodiments, the plasmid comprising the recombinant polynucleotide is SEQ ID NO: 50. In some embodiments, the recombinant polynucleotide has at least 60%, 65%, 70% 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence comprising the recombinant polynucleotide between the ITRs of SEQ ID NO: 50.

[0255] Another example of plasmid for expressing progranulin is SEQ ID NO: 51. The plasmid of SEQ ID NO: 51 contains inverted terminal repeats flanking a sequence encoding a CMV promoter, a sequence encoding progranulin, a WPRE, and a bGH polyA signal. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 80% sequence identity to SEQ ID NO: 51. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 85% sequence identity to SEQ ID NO: 51. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 90% sequence identity to SEQ ID NO: 51. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 95% sequence identity to SEQ ID NO: 51. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 96% sequence identity to SEQ ID NO: 51. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 97% sequence identity to SEQ ID NO: 51. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 98% sequence identity to SEQ ID NO: 51. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 99% sequence identity to SEQ ID NO: 51. In some embodiments, the plasmid comprising the recombinant polynucleotide comprises 100% sequence identity to SEQ ID NO: 51. In some embodiments, the plasmid comprising the recombinant polynucleotide is SEQ ID NO: 51. In some embodiments, the recombinant polynucleotide has at least 60%, 65%, 70% 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence comprising the recombinant polynucleotide between the ITRs of SEQ ID NO: 51.

[0256] Another example of plasmid for expressing progranulin is SEQ ID NO: 52. The plasmid of SEQ ID NO: 52 contains inverted terminal repeats flanking a sequence encoding a hSyn promoter, a sequence encoding progranulin, a WPRE, and a beta globin polyA signal. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 80% sequence identity to SEQ ID NO: 52. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 85% sequence identity to SEQ ID NO: 52. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 90% sequence identity to SEQ ID NO: 52. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 95% sequence identity to SEQ ID NO: 52. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 96% sequence identity to SEQ ID NO: 52. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 97% sequence identity to SEQ ID NO: 52. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 98% sequence identity to SEQ ID NO: 52. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 99% sequence identity to SEQ ID NO: 52. In some embodiments, the plasmid comprising the recombinant polynucleotide comprises 100% sequence identity to SEQ ID NO: 52. In some embodiments, the plasmid comprising the recombinant polynucleotide is SEQ ID NO: 52. In some embodiments, the recombinant polynucleotide has at least 60%, 65%, 70% 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence comprising the recombinant polynucleotide between the ITRs of SEQ ID NO: 52.

[0257] Another example of plasmid for expressing progranulin is SEQ ID NO: 53. The plasmid of SEQ ID NO: 53 contains inverted terminal repeats flanking a sequence encoding an RSV promoter, a sequence encoding progranulin, and a rabbit beta globin polyA signal. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 80% sequence identity to SEQ ID NO: 53. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 85% sequence identity to SEQ ID NO: 53. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 90% sequence identity to SEQ ID NO: 53. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 95% sequence identity to SEQ ID NO: 53. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 96% sequence identity to SEQ ID NO: 53. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 97% sequence identity to SEQ ID NO: 53. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 98% sequence identity to SEQ ID NO: 53. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 99% sequence identity to SEQ ID NO: 53. In some embodiments, the plasmid comprising the recombinant polynucleotide comprises 100% sequence identity to SEQ ID NO: 53. In some embodiments, the plasmid comprising the recombinant polynucleotide is SEQ ID NO: 53. In some embodiments, the recombinant polynucleotide has at least 60%, 65%, 70% 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence comprising the recombinant polynucleotide between the ITRs of SEQ ID NO: 53.

[0258] Another example of plasmid for expressing progranulin is SEQ ID NO: 71. The plasmid of SEQ ID NO: 71 contains inverted terminal repeats flanking a sequence encoding an RSV promoter and a sequence encoding progranulin. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 80% sequence identity to SEQ ID NO: 71. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 85% sequence identity to SEQ ID NO: 71. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 90% sequence identity to SEQ ID NO: 71. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 95% sequence identity to SEQ ID NO: 71. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 96% sequence identity to SEQ ID NO: 71. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 97% sequence identity to SEQ ID NO: 71. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 98% sequence identity to SEQ ID NO: 71. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 99% sequence identity to SEQ ID NO: 71. In some embodiments, the plasmid comprising the recombinant polynucleotide comprises 100% sequence identity to SEQ ID NO: 71. In some embodiments, the plasmid comprising the recombinant polynucleotide is SEQ ID NO: 71. In some embodiments, the recombinant polynucleotide has at least 60%, 65%, 70% 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence comprising the recombinant polynucleotide between the ITRs of SEQ ID NO: 71.

[0259] Another example of plasmid for expressing progranulin is SEQ ID NO: 72. The plasmid of SEQ ID NO: 72 contains inverted terminal repeats flanking a sequence encoding a JeT promoter and a sequence encoding progranulin. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 80% sequence identity to SEQ ID NO: 72. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 85% sequence identity to SEQ ID NO: 72. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 90% sequence identity to SEQ ID NO: 72. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 95% sequence identity to SEQ ID NO: 72. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 96% sequence identity to SEQ ID NO: 72. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 97% sequence identity to SEQ ID NO: 72. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 98% sequence identity to SEQ ID NO: 72. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 99% sequence identity to SEQ ID NO: 72. In some embodiments, the plasmid comprising the recombinant polynucleotide comprises 100% sequence identity to SEQ ID NO: 72. In some embodiments, the plasmid comprising the recombinant polynucleotide is SEQ ID NO: 72. In some embodiments, the recombinant polynucleotide has at least 60%, 65%, 70% 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence comprising the recombinant polynucleotide between the ITRs of SEQ ID NO: 72.

[0260] Another example of plasmid for expressing progranulin is SEQ ID NO: 73. The plasmid of SEQ ID NO: 73 contains inverted terminal repeats flanking a sequence encoding an RSV promoter and a sequence encoding progranulin including GRN intron 9. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 80% sequence identity to SEQ ID NO: 73. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 85% sequence identity to SEQ ID NO: 73. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 90% sequence identity to SEQ ID NO: 73. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 95% sequence identity to SEQ ID NO: 73. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 96% sequence identity to SEQ ID NO: 73. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 97% sequence identity to SEQ ID NO: 73. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 98% sequence identity to SEQ ID NO: 73. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 99% sequence identity to SEQ ID NO: 73. In some embodiments, the plasmid comprising the recombinant polynucleotide comprises 100% sequence identity to SEQ ID NO: 73. In some embodiments, the plasmid comprising the recombinant polynucleotide is SEQ ID NO: 73. In some embodiments, the recombinant polynucleotide has at least 60%, 65%, 70% 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence comprising the recombinant polynucleotide between the ITRs of SEQ ID NO: 73.

[0261] Another example of plasmid for expressing progranulin is SEQ ID NO: 74. The plasmid of SEQ ID NO: 74 contains inverted terminal repeats flanking a sequence encoding an RSV promoter, an SV40 intron, and a sequence encoding progranulin. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 80% sequence identity to SEQ ID NO: 74. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 85% sequence identity to SEQ ID NO: 74. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 90% sequence identity to SEQ ID NO: 74. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 95% sequence identity to SEQ ID NO: 74. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 96% sequence identity to SEQ ID NO: 74. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 97% sequence identity to SEQ ID NO: 74. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 98% sequence identity to SEQ ID NO: 74. In some embodiments, the plasmid comprising the recombinant polynucleotide has at least 99% sequence identity to SEQ ID NO: 74. In some embodiments, the plasmid comprising the recombinant polynucleotide comprises 100% sequence identity to SEQ ID NO: 74. In some embodiments, the plasmid comprising the recombinant polynucleotide is SEQ ID NO: 74. In some embodiments, the recombinant polynucleotide has at least 60%, 65%, 70% 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence comprising the recombinant polynucleotide between the ITRs of SEQ ID NO: 74.

[0262] In some examples, the delivery vector can be a retroviral vector. In some examples, the retroviral vector can be a Moloney Murine Leukemia Virus vector, a spleen necrosis virus vector, or a vector derived from the Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, or mammary tumor virus, or a combination thereof. In some examples, the retroviral vector can be transfected such that the majority of sequences coding for the structural genes of the virus (e.g., gag, pol, and env) can be deleted and replaced by the gene(s) of interest.

[0263] In some examples, the delivery vehicle is a non- viral vector. Examples of non- viral vectors include plasmids, lipid nanoparticles, lipoplexes, polymersomes, polyplexes, dendrimers, nanoparticles, and cell-penetrating peptides. The non-viral vector can comprise a polynucleotide, such as a plasmid, encoding for a promoter (e.g., comprising a cell type- or cell state-specific response element and a switchable core promoter) and a payload sequence. In some examples, the delivery vehicle is a plasmid. In some examples, the plasmid is a minicircle plasmid. In some embodiments, a vector comprises naked DNA (e.g., a naked DNA plasmid). In some embodiments, the non-viral vector comprises DNA. In some embodiments, the non-viral vector comprises RNA. In some examples, the non-viral vector comprises circular doublestranded DNA. In some examples, the non-viral vector comprises a linear polynucleotide. In some examples, the non-viral vector comprises a polynucleotide encoding one or more genes of interest and one or more regulatory elements. In some examples, the non-viral vector comprises a bacterial backbone containing an origin of replication and an antibiotic resistance gene or other selectable marker for plasmid amplification in bacteria. In some examples, the non-viral vector contains one or more genes that provide a selective marker to induce a target cell to retain a polynucleotide (e.g., a plasmid) of the non-viral vector. In some examples, the non-viral vector is formulated for delivery through injection by a needle carrying syringe. In some examples, the non-viral vector is formulated for delivery via electroporation. In some examples, a polynucleotide of the non-viral vector is engineered through synthetic or other suitable means known in the art. For example, in some cases, the genetic elements can be assembled by restriction digest of the desired genetic sequence from a donor plasmid or organism to produce ends of the DNA which can then be readily ligated to another genetic sequence.

[0264] In some embodiments, the vector containing the recombinant polynucleotide is a non- viral vector system. In some embodiments, the non-viral vector system comprises cationic lipids, or polymers. For example, the non-viral vector system comprises can be a liposome or polymeric nanoparticle. In some embodiments, the recombinant polynucleotide or a non-viral vector comprising the recombinant polynucleotide is delivered to a cell by hydrodynamic injection or ultrasound.

Pharmaceutical Compositions

[0265] Methods for treatment of diseases or disorders characterized by genetic mutations or aberrant gene expression are also encompassed by the present disclosure. Said methods include administering a therapeutically effective amount of a payload sequence as part of a recombinant polynucleotide. The recombinant polynucleotide of the disclosure can be formulated in pharmaceutical compositions. These compositions can comprise, in addition to one or more of the recombinant polynucleotides, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be nontoxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can depend on the route of administration, e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.

[0266] The compositions described herein (e.g., compositions comprising an engineered guide RNA or an engineered polynucleotide) can be formulated with a pharmaceutically acceptable carrier for administration to a subject (e.g., a human or a non-human animal). A pharmaceutically acceptable carrier can include, but is not limited to, phosphate buffered saline solution, water, emulsions (e.g., an oil/water emulsion or a water/oil emulsions), glycerol, liquid polyethylene glycols, aprotic solvents such (e.g., dimethylsulfoxide, N-methylpyrrolidone, or mixtures thereof), and various types of wetting agents, solubilizing agents, anti-oxidants, bulking agents, protein carriers such as albumins, any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintegrants (e.g., potato starch or sodium starch glycolate), and the like. The compositions also can include stabilizers and preservatives. Additional examples of carriers, stabilizers and adjuvants consistent with the compositions of the present disclosure can be found in, for example, Remington's Pharmaceutical Sciences, 21st Ed., Mack Publ. Co., Easton, Pa. (2005), incorporated herein by reference in its entirety.

[0267] In some examples, the pharmaceutical composition can be formulated in unit dose forms or multiple-dose forms. In some examples, the unit dose forms can be physically discrete units suitable for administration to human or non-human subjects (e.g., animals). In some examples, the unit dose forms can be packaged individually. In some examples, each unit dose contains a predetermined quantity of an active ingredient(s) that can be sufficient to produce the desired therapeutic effect in association with pharmaceutical carriers, diluents, excipients, or any combination thereof. In some examples, the unit dose forms comprise ampules, syringes, or individually packaged tablets and capsules, or any combination thereof. In some instances, a unit dose form can be comprised in a disposable syringe. In some instances, unit-dosage forms can be administered in fractions or multiples thereof. In some examples, a multiple-dose form comprises a plurality of identical unit dose forms packaged in a single container, which can be administered in segregated a unit dose form. In some examples, multiple dose forms comprise vials, bottles of tablets or capsules, or bottles of pints or gallons. In some instances, a multipledose forms comprise the same pharmaceutically active agents. In some instances, a multipledose forms comprise different pharmaceutically active agents.

[0268] In some examples, the pharmaceutical composition comprises a pharmaceutically acceptable excipient. In some examples, the excipient comprises a buffering agent, a cryopreservative, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a chelator, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, or a coloring agent, or any combination thereof.

[0269] In some examples, an excipient comprises a buffering agent. In some examples, the buffering agent comprises sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, calcium bicarbonate, or any combination thereof. In some examples, the buffering agent comprises sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium glucomate, aluminum hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, calcium acetate, calcium glycerophosphate, calcium chloride, or calcium hydroxide and other calcium salts, or any combination thereof.

[0270] In some examples, an excipient comprises a cryopreservative. In some examples, the cryopreservative comprises DMSO, glycerol, polyvinylpyrrolidone (PVP), or any combination thereof. In some examples, a cryopreservative comprises a sucrose, a trehalose, a starch, a salt of any of these, a derivative of any of these, or any combination thereof. In some examples, an excipient comprises a pH agent (to minimize oxidation or degradation of a component of the composition), a stabilizing agent (to prevent modification or degradation of a component of the composition), a buffering agent (to enhance temperature stability), a solubilizing agent (to increase protein solubility), or any combination thereof. In some examples, an excipient comprises a surfactant, a sugar, an amino acid, an antioxidant, a salt, a non-ionic surfactant, a solubilizer, a triglyceride, an alcohol, or any combination thereof. In some examples, an excipient comprises sodium carbonate, acetate, citrate, phosphate, poly-ethylene glycol (PEG), human serum albumin (HSA), sorbitol, sucrose, trehalose, polysorbate 80, sodium phosphate, sucrose, disodium phosphate, mannitol, polysorbate 20, histidine, citrate, albumin, sodium hydroxide, glycine, sodium citrate, trehalose, arginine, sodium acetate, acetate, HC1, disodium edetate, lecithin, glycerin, xanthan rubber, soy isoflavones, polysorbate 80, ethyl alcohol, water, teprenone, or any combination thereof. In some examples, the excipient can be an excipient described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986).

[0271] In some examples, the excipient comprises a preservative. In some examples, the preservative comprises an antioxidant, such as alpha-tocopherol and ascorbate, an antimicrobial, such as parabens, chlorobutanol, and phenol, or any combination thereof. In some examples, the antioxidant comprises EDTA, citric acid, ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxy anisole (BHA), sodium sulfite, p-amino benzoic acid, glutathione, propyl gallate, cysteine, methionine, ethanol or N- acetyl cysteine, or any combination thereof. In some examples, the preservative comprises validamycin A, TL-3, sodium ortho vanadate, sodium fluoride, N-a-tosyl-Phe- chloromethylketone, N-a-tosyl-Lys-chloromethylketone, aprotinin, phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, kinase inhibitor, phosphatase inhibitor, caspase inhibitor, granzyme inhibitor, cell adhesion inhibitor, cell division inhibitor, cell cycle inhibitor, lipid signaling inhibitor, protease inhibitor, reducing agent, alkylating agent, antimicrobial agent, oxidase inhibitor, or other inhibitors, or any combination thereof.

[0272] In some examples, the excipient comprises a binder. In some examples, the binder comprises starches, pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, or any combination thereof.

[0273] In some examples, the binder can be a starch, for example a potato starch, corn starch, or wheat starch; a sugar such as sucrose, glucose, dextrose, lactose, or maltodextrin; a natural and/or synthetic gum; a gelatin; a cellulose derivative such as microcrystalline cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, methyl cellulose, or ethyl cellulose; polyvinylpyrrolidone (povidone); polyethylene glycol (PEG); a wax; calcium carbonate; calcium phosphate; an alcohol such as sorbitol, xylitol, mannitol, or water, or any combination thereof.

[0274] In some examples, the excipient comprises a lubricant. In some examples, the lubricant comprises magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils, sterotex, polyoxyethylene monostearate, talc, polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, or light mineral oil, or any combination thereof. In some examples, the lubricant comprises metallic stearates (such as magnesium stearate, calcium stearate, aluminum stearate), fatty acid esters (such as sodium stearyl fumarate), fatty acids (such as stearic acid), fatty alcohols, glyceryl behenate, mineral oil, paraffins, hydrogenated vegetable oils, leucine, polyethylene glycols (PEG), metallic lauryl sulphates (such as sodium lauryl sulphate, magnesium lauryl sulphate), sodium chloride, sodium benzoate, sodium acetate or talc or a combination thereof.

[0275] In some examples, the excipient comprises a dispersion enhancer. In some examples, the dispersion enhancer comprises starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isomorphous silicate, or microcrystalline cellulose, or any combination thereof as high HLB emulsifier surfactants.

[0276] In some examples, the excipient comprises a disintegrant. In some examples, a disintegrant comprises a non-effervescent disintegrant. In some examples, a non-effervescent disintegrants comprises starches such as com starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro-crystalline cellulose, alginates, sodium starch glycolate, or gums such as agar, guar, locust bean, karaya, pectin, and tragacanth, or any combination thereof. In some examples, a disintegrant comprises an effervescent disintegrant. In some examples, a suitable effervescent disintegrant comprises bicarbonate in combination with citric acid, and sodium bicarbonate in combination with tartaric acid.

[0277] In some examples, the excipient comprises a sweetener, a flavoring agent or both. In some exmaples, a sweetener comprises glucose (com syrup), dextrose, invert sugar, fructose, and mixtures thereof (when not used as a carrier); saccharin and its various salts such as a sodium salt; dipeptide sweeteners such as aspartame; dihydrochalcone compounds, glycyrrhizin; Stevia Rebaudiana (Stevioside); chloro derivatives of sucrose such as sucralose; and sugar alcohols such as sorbitol, mannitol, sylitol, and the like, or any combination thereof. In some cases, flavoring agents incorporated into a composition comprise synthetic flavor oils and flavoring aromatics; natural oils; extracts from plants, leaves, flowers, and fruits; or any combination thereof. In some embodiments, a flavoring agent comprises a cinnamon oils; oil of wintergreen; peppermint oils; clover oil; hay oil; anise oil; eucalyptus; vanilla; citrus oil such as lemon oil, orange oil, grape and grapefruit oil; and fruit essences including apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple, and apricot, or any combination thereof.

[0278] In some examples, the excipient comprises a pH agent (e.g., to minimize oxidation or degradation of a component of the composition), a stabilizing agent (e.g., to prevent modification or degradation of a component of the composition), a buffering agent (e.g., to enhance temperature stability), a solubilizing agent (e.g., to increase protein solubility), or any combination thereof. In some examples, the excipient comprises a surfactant, a sugar, an amino acid, an antioxidant, a salt, a non-ionic surfactant, a solubilizer, a trigylceride, an alcohol, or any combination thereof. In some examples, the excipient comprises sodium carbonate, acetate, citrate, phosphate, poly-ethylene glycol (PEG), human serum albumin (HSA), sorbitol, sucrose, trehalose, polysorbate 80, sodium phosphate, sucrose, disodium phosphate, mannitol, polysorbate 20, histidine, citrate, albumin, sodium hydroxide, glycine, sodium citrate, trehalose, arginine, sodium acetate, acetate, HC1, disodium edetate, lecithin, glycerine, xanthan rubber, soy isoflavones, polysorbate 80, ethyl alcohol, water, teprenone, or any combination thereof. In some examples, the excipient comprises a cryo-preservative. In some examples, the excipient comprises DMSO, glycerol, polyvinylpyrrolidone (PVP), or any combination thereof. In some examples, the excipient comprises a sucrose, a trehalose, a starch, a salt of any of these, a derivative of any of these, or any combination thereof.

[0279] In some examples, the pharmaceutical composition comprises a diluent. In some examples, the diluent comprises water, glycerol, methanol, ethanol, or other similar biocompatible diluents, or any combination thereof. In some examples, a diluent comprises an aqueous acid such as acetic acid, citric acid, maleic acid, hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid, or any combination thereof. In some examples, a diluent comprises an alkaline metal carbonates such as calcium carbonate; alkaline metal phosphates such as calcium phosphate; alkaline metal sulphates such as calcium sulphate; cellulose derivatives such as cellulose, microcrystalline cellulose, cellulose acetate; magnesium oxide, dextrin, fructose, dextrose, glyceryl palmitostearate, lactitol, choline, lactose, maltose, mannitol, simethicone, sorbitol, starch, pregelatinized starch, talc, xylitol and/or anhydrates, hydrates and/or pharmaceutically acceptable derivatives thereof or combinations thereof.

[0280] In some examples, the pharmaceutical composition comprises a carrier. In some examples, the carrier comprises a liquid or solid filler, solvent, or encapsulating material. In some examples, the carrier comprises additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-oligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldolic acids, esterified sugars and the like; and polysaccharides or sugar polymers), alone or in combination.

Administration

[0281] Administration can refer to methods that can be used to enable the delivery of a composition described herein (e.g., comprising an engineered guide RNA or an engineered polynucleotide encoding the same) to the desired site of biological action. For example, an recombinant polynucleotide can be comprised in a DNA construct, a viral vector, or both and be administered by intravenous administration. Administration disclosed herein to an area in need of treatment or therapy can be achieved by, for example, and not by way of limitation, oral administration, topical administration, intravenous administration, inhalation administration, or any combination thereof. In some embodiments, delivery can include inhalation, otic, buccal, conjunctival, dental, endocervical, endosinusial, endotracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration, interstitial, intraabdominal, intraamniotic, intraarterial, intraarticular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavemous, intracavitary, intracerebroventricular, intracranial ventricular (ICV), intracistemal, intracorneal, intracoronal, intracoronary, intracorpous cavernaosum, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intrahippocampal, intraileal, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intravenous, intravenous bolus, intravenous drip, intravesical, intravitreal, iontophoresis, irrigation, laryngeal, nasal, nasogastric, ophthalmic, oral, oropharyngeal, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, retrobulbar, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanic, ureteral, urethral, vaginal, infraorbital, intraparenchymal, intrathecal, intraventricular, stereotactic, or any combination thereof. Delivery can include parenteral administration (including intravenous, subcutaneous, intrathecal, intraperitoneal, intramuscular, intravascular or infusion), oral administration, inhalation administration, intraduodenal administration, rectal administration, or a combination thereof. Delivery can include direct application to the affected tissue or region of the body. In some cases, topical administration can comprise administering a lotion, a solution, an emulsion, a cream, a balm, an oil, a paste, a stick, an aerosol, a foam, a jelly, a foam, a mask, a pad, a powder, a solid, a tincture, a butter, a patch, a gel, a spray, a drip, a liquid formulation, an ointment to an external surface of a surface, such as a skin. Delivery can include a parenchymal injection, an intra-thecal injection, an intra- ventricular injection, or an intra-cisternal injection. A composition provided herein can be administered by any method. A method of administration can be by intra-arterial injection, intracisternal injection, intramuscular injection, intraparenchymal injection, intraperitoneal injection, intraspinal injection, intrathecal injection, intravenous injection, intraventricular injection, stereotactic injection, subcutaneous injection, epidural, or any combination thereof. In some embodiments, a preferred method of administration is intravenous administration. Delivery can include parenteral administration (including intravenous, subcutaneous, intrathecal, intraperitoneal, intramuscular, intravascular, or infusion administration). In some embodiments, delivery can comprise a nanoparticle, a liposome, an exosome, an extracellular vesicle, an implant, or a combination thereof. In some cases, delivery can be from a device. In some instances, delivery can be administered by a pump, an infusion pump, or a combination thereof. In some embodiments, delivery can be by an enema, an eye drop, a nasal spray, or any combination thereof. In some instances, a subject can administer the composition in the absence of supervision. In some instances, a subject can administer the composition under the supervision of a medical professional (e.g., a physician, nurse, physician’s assistant, orderly, hospice worker, etc.). In some embodiments, a medical professional can administer the composition.

[0282] In some cases, administering can be oral ingestion. In some cases, delivery can be a capsule or a tablet. Oral ingestion delivery can comprise a tea, an elixir, a food, a drink, a beverage, a syrup, a liquid, a gel, a capsule, a tablet, an oil, a tincture, or any combination thereof. In some embodiments, a food can be a medical food. In some instances, a capsule can comprise hydroxymethylcellulose. In some embodiments, a capsule can comprise a gelatin, hydroxypropylmethyl cellulose, pullulan, or any combination thereof. In some cases, capsules can comprise a coating, for example, an enteric coating. In some embodiments, a capsule can comprise a vegetarian product or a vegan product such as a hypromellose capsule. In some embodiments, delivery can comprise inhalation by an inhaler, a diffuser, a nebulizer, a vaporizer, or a combination thereof.

[0283] In some embodiments, disclosed herein can be a method, comprising administering a composition disclosed herein to a subject (e.g., a human) in need thereof. In some instances, the method can treat (including prevent) a disease in the subject.

[0284] In some examples, a pharmaceutical composition disclosed herein can be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, or prophylactic, effect.

[0285] The appropriate dosage and treatment regimen for the methods of treatment described herein vary with respect to the particular disease being treated, the recombinant polynucleotide being delivered, and the specific condition of the subject. In some examples, the administration can be over a period of time until the desired effect (e.g., reduction in symptoms can be achieved). In some examples, administration can be 1, 2, 3, 4, 5, 6, or 7 times per week. In some examples, administration or application of a composition disclosed herein can be performed for a treatment duration of at least about 1 week, at least about 1 month, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 15 years, at least about 20 years, or more. In some examples, administration can be over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In some examples, administration can be over a period of 2, 3, 4, 5, 6 or more months. In some examples, administration can be performed repeatedly over a lifetime of a subject, such as once a month or once a year for the lifetime of a subject. In some examples, administration can be performed repeatedly over a substantial portion of a subject’s life, such as once a month or once a year for at least about 1 year, 5 years, 10 years, 15 years, 20 years, 25 years, 30 years, or more. In some examples, treatment can be resumed following a period of remission. [0286] Pharmaceutical compositions for oral administration can be in tablet, capsule, powder, or liquid form. A tablet can include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be included.

[0287] For intravenous, ICV, cutaneous, or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen- free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives can be included, as required.

[0288] In some embodiments, the recombinant polynucleotide of the present disclosure is administered to cells via a lipid nanoparticle. In some embodiments, the lipid nanoparticle is administered at the appropriate concentration according to standard methods appropriate for the target cells.

[0289] In some embodiments, the recombinant polynucleotide of the present disclosure are administered to cells via a viral vector. In some embodiments, the viral vector is administered at the appropriate multiplicity of infection according to standard transduction methods appropriate for the target cells. Titers of the virus vector or capsid to administer can vary depending on the target cell type or cell state and number and can be determined by those of skill in the art. In some embodiments, at least about 10² infections units are administered. In some embodiments, at least about 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, IO¹⁰, 10¹¹, 10¹², or 10¹³ infectious units are administered.

[0290] In some embodiments, the recombinant polynucleotide is introduced to cells of any type or state, including, but not limited to neural cells, cells of the eye (including retinal cells, retinal pigment epithelium, and comeal cells), lung cells, epithelial cells, skeletal muscle cells, dendritic cells, hepatic cells, pancreatic cells, bone cells, hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, and heart cells.

[0291] In some embodiments, the recombinant polynucleotide of the disclosure is introduced to cells in vitro via a viral vector for administration of modified cells to a subject. In some embodiments, a viral vector encoding the recombinant polynucleotide of the disclosure is introduced to cells that have been removed from a subject. In some embodiments, the modified cells are placed back in the subject following introduction of the viral vector. [0292] In some embodiments, a dose of modified cells is administered to a subject according to the age and species of the subject, disease or disorder to be treated, as well as the cell type or state and mode of administration. In some embodiments, at least about 10² - 10⁸ cells are administered per dose. In some embodiments, cells transduced with viral vector are administered to a subject in an effective amount.

[0293] In some embodiments, the dose of viral vector administered to a subject will vary according to the age of the subject, the disease or disorder to be treated, and mode of administration. In some embodiments, the dose for achieving a therapeutic effect is a virus titer of at least about 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, IO¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶ or more transducing units.

[0294] Administration of the pharmaceutically useful the recombinant polynucleotide of the present disclosure is preferably in a “therapeutically effective amount” or “prophylactically effective amount” (as the case can be, although prophylaxis can be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of protein aggregation disease being treated. Prescription of treatment, e.g., decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0295] A composition can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

[0296] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein is intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0297] The term “complementary” or “complementarity” refers to the ability of a nucleic acid to form one or more bonds with a corresponding nucleic acid sequence by, for example, hydrogen bonding (e.g., traditional Watson-Crick), covalent bonding, or other similar methods. In Watson- Crick base pairing, a double hydrogen bond forms between nucleobases T and A, whereas a triple hydrogen bond forms between nucleobases C and G. For example, the sequence A-G-T can be complementary to the sequence T-C-A. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson- Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). “Perfectly complementary” can mean that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein can refer to a degree of complementarity that can be at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%. 97%, 98%, 99%, or 100% over a region of 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides, or can refer to two nucleic acids that hybridize under stringent conditions (i.e., stringent hybridization conditions). Nucleic acids can include nonspecific sequences. As used herein, the term “nonspecific sequence” or “not specific” can refer to a nucleic acid sequence that contains a series of residues that can be not designed to be complementary to or can be only partially complementary to any other nucleic acid sequence.

[0298] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” can be used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative, or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0299] The term “encode,” as used herein, refers to an ability of a polynucleotide to provide information or instructions sequence sufficient to produce a corresponding gene expression product. In a non-limiting example, mRNA can encode a polypeptide during translation, whereas DNA can encode an mRNA molecule during transcription.

[0300] “Messenger RNA” or “mRNA” are RNA molecules comprising a sequence that encodes a polypeptide or protein. In general, RNA can be transcribed from DNA. In some cases, precursor mRNA containing non-protein coding regions in the sequence can be transcribed from DNA and then processed to remove all or a portion of the non-coding regions (introns) to produce mature mRNA. As used herein, the term “pre-mRNA” can refer to the RNA molecule transcribed from DNA before undergoing processing to remove the non-protein coding regions. [0301] The terms “subject,” “individual,” or “patient” can be used interchangeably herein. A “subject” refers to a biological entity containing expressed genetic materials. The biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. The subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. The subject can be a mammal. The mammal can be a human. The subject can be diagnosed or suspected of being at high risk for a disease. In some cases, the subject may not be necessarily diagnosed or suspected of being at high risk for the disease [0302] The term “zh vivo” refers to an event that takes place in a subject’s body.

[0303] The term “ex vivo” refers to an event that takes place outside of a subject’s body. An ex vivo assay may not be performed on a subject. Rather, it can be performed upon a sample separate from a subject. An example of an ex vivo assay performed on a sample can be an “zh vitro” assay.

[0304] The term “zh vitro” refers to an event that takes places contained in a container for holding laboratory reagent such that it can be separated from the biological source from which the material can be obtained. In vitro assays can encompass cell-based assays in which living or dead cells can be employed. In vitro assays can also encompass a cell-free assay in which no intact cells can be employed.

[0305] As used herein, the term “polynucleotide” refers to a single or double-stranded polymer of deoxyribonucleotide (DNA) or ribonucleotide (RNA) bases read from the 5 ’ to the 3 ’ end. The term “RNA” is inclusive of dsRNA (double stranded RNA), snRNA (small nuclear RNA), IncRNA (long non-coding RNA), mRNA (messenger RNA), miRNA (microRNA) RNAi (inhibitory RNA), siRNA (small interfering RNA), shRNA (short hairpin RNA), tRNA (transfer RNA), rRNA (ribosomal RNA), snoRNA (small nucleolar RNA), and cRNA (complementary RNA). The term DNA is inclusive of cDNA, genomic DNA, and DNA-RNA hybrids. A sequence of a polynucleotide may be provided interchangeably as an RNA sequence (containing U) or a DNA sequence (containing T). A sequence provided as an RNA sequence is intended to also cover the corresponding DNA sequence and the reverse complement RNA sequence or DNA sequence. A sequence provided as a DNA sequence is intended to also cover the corresponding RNA sequence and the reverse complement RNA sequence or DNA sequence. [0306] The term “protein”, “peptide” and “polypeptide” can be used interchangeably and in their broadest sense can refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits can be linked by peptide bonds. In another embodiment, the subunit can be linked by other bonds, e.g., ester, ether, etc. A protein or peptide can contain at least two amino acids and no limitation can be placed on the maximum number of amino acids which can comprise a protein’s or peptide's sequence. As used herein the term “amino acid” can refer to either natural amino acids, unnatural amino acids, or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics. As used herein, the term “fusion protein” can refer to a protein comprised of domains from more than one naturally occurring or recombinantly produced protein, where generally each domain serves a different function. In this regard, the term “linker” can refer to a protein fragment that can be used to link these domains together - optionally to preserve the conformation of the fused protein domains, prevent unfavorable interactions between the fused protein domains which can compromise their respective functions, or both.

[0307] The term “ameliorating” refers to any therapeutically beneficial result in the treatment of a disease state, e.g., Rett syndrome, including prophylaxis, lessening in the severity or progression, remission, or cure thereof.

[0308] The term “mammal” as used herein includes both humans and non-humans and include but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0309] The term percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

[0310] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0311] Unless otherwise stated, whenever a range is recited, the range is inclusive of the recited endpoints. For example, the region from amino acid residue 581 to amino acid residue 589 of SEQ ID NO: 1 includes amino acid residues 581 and 589.

[0312] The term percent “identity” or “homology,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

[0313] For sequence comparison, typically one sequence acts as a reference sequence (also called the subject sequence) to which test sequences (also called query sequences) are compared. The percent sequence identity is defined as a test sequence’s percent identity to a reference sequence. For example, when stated “Sequence A having a sequence identity of 50% to Sequence B,” Sequence A is the test sequence and Sequence B is the reference sequence. When using a sequence comparison algorithm, test and reference sequences are input into a computer program, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then aligns the sequences to achieve the maximum alignment, based on the designated program parameters, introducing gaps in the alignment if necessary. The percent sequence identity for the test sequence(s) relative to the reference sequence can then be determined from the alignment of the test sequence to the reference sequence. The equation for percent sequence identity from the aligned sequence is as follows:

[(Number of Identical Positions)/(Total Number of Positions in the Test Sequence)] x 100%

[0314] For purposes herein, percent identity and sequence similarity calculations are performed using the BLAST algorithm for sequence alignment, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/). The BLAST algorithm uses a test sequence (also called a query sequence) and a reference sequence (also called a subject sequence) to search against, or in some cases, a database of multiple reference sequences to search against. The BLAST algorithm performs sequence alignment by finding high-scoring alignment regions between the test and the reference sequences by scoring alignment of short regions of the test sequence (termed “words”) to the reference sequence. The scoring of each alignment is determined by the BLAST algorithm and takes factors into account, such as the number of aligned positions, as well as whether introduction of gaps between the test and the reference sequences would improve the alignment. The alignment scores for nucleic acids can be scored by set match/mismatch scores. For protein sequences, the alignment scores can be scored using a substitution matrix to evaluate the significance of the sequence alignment, for example, the similarity between aligned amino acids based on their evolutionary probability of substitution. For purposes herein, the substitution matrix used is the BLOSUM62 matrix. For purposes herein, the public default values of April 6, 2023 are used when using the BLASTN and BLASTP algorithms. The BLASTN and BLASTP algorithms then output a “Percent Identity” output value and a “Query Coverage” output value. The overall percent sequence identity as used herein can then be calculated from the BLASTN or BLASTP output values as follows:

Percent Sequence Identity = (“Percent Identity” output value) x (“Query Coverage” output value)

[0315] The following non-limiting examples illustrate the calculation of percent identity between two nucleic acids sequences. The percent identity is calculated as follows: [(number of identical nucleotide positions)/(total number of nucleotides in the test sequence)] x 100%. Percent identity is calculated to compare test sequence 1: AAAAAGGGGG (SEQ ID NO: 112; length = 10 nucleotides) to reference sequence 2: AAAAAAAAAA (SEQ ID NO: 113; length = 10 nucleotides). The percent identity between test sequence 1 and reference sequence 2 would be [(5)/(10)] xioo% = 50%. Test sequence 1 has 50% sequence identity to reference sequence 2. In another example, percent identity is calculated to compare test sequence 3: CCCCCGGGGGGGGGGCCCCC (SEQ ID NO: 114; length = 20 nucleotides) to reference sequence 4: GGGGGGGGGG (SEQ ID NO: 115; length = 10 nucleotides). The percent identity between test sequence 3 and reference sequence 4 would be [(10)/(20)] xl00% = 50%. Test sequence 3 has 50% sequence identity to reference sequence 4. In another example, percent identity is calculated to compare test sequence 5: GGGGGGGGGG (SEQ ID NO: 115; length = 10 nucleotides) to reference sequence 6: CCCCCGGGGGGGGGGCCCCC (SEQ ID NO: 114; length = 20 nucleotides). The percent identity between test sequence 5 and reference sequence 6 would be [(10)/(10)] xioo% = 100%. Test sequence 5 has 100% sequence identity to reference sequence 6.

[0316] The following non-limiting examples illustrate the calculation of percent identity between two protein sequences. The percent identity is calculated as follows: [(number of identical amino acid positions)/(total number of amino acids in the test sequence)] x 100%. Percent identity is calculated to compare test sequence 7: FFFFFYYYYY (SEQ ID NO: 116; length = 10 amino acids) to reference sequence 8: YYYYYYYYYY (SEQ ID NO: 117; length = 10 amino acids). The percent identity between test sequence 7 and reference sequence 8 would be [(5)/(10)] xioo% = 50%. Test sequence 7 has 50% sequence identity to reference sequence 8. In another example, percent identity is calculated to compare test sequence 9: LLLLLFFFFFYYYYYLLLLL (SEQ ID NO: 118; length = 20 amino acids) to reference sequence 10: FFFFFYYYYY (SEQ ID NO: 116; length = 10 amino acids). The percent identity between test sequence 9 and reference sequence 10 would be [(10)/(20)] >< 100% = 50%. Test sequence 9 has 50% sequence identity to reference sequence 10. In another example, percent identity is calculated to compare test sequence 11: FFFFFYYYYY (SEQ ID NO: 116; length = 10 amino acids) to reference sequence 12: LLLLLFFFFFYYYYYLLLLL (SEQ ID NO: 118; length = 20 amino acids). The percent identity between test sequence 11 and reference sequence 12 would be [(10)/(l 0)] ><100% = 100%. Test sequence 11 has 100% sequence identity to reference sequence 12.

[0317] For purposes herein, reference to a polynucleotide sequence (e.g., a DNA sequence or an RNA sequence) also encompasses the reverse complement of the polynucleotide sequence. For example, a sequence of AAAAAGGGGG (SEQ ID NO: 112) also encompasses a sequence of CCCCCTTTTT (SEQ ID NO: 119).

[0318] As used herein, the term “subject” broadly refers to any animal, including but not limited to, human and non-human animals (e.g., dogs, cats, cows, horses, sheep, pigs, poultry, fish, crustaceans, etc.).

[0319] As used herein, the term “effective amount” refers to the amount of a composition (e.g., a synthetic peptide) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.

[0320] As used herein, the term “therapeutically effective amount” is an amount that is effective to ameliorate a symptom of a disease. A therapeutically effective amount can be a “prophylactically effective amount” as prophylaxis can be considered therapy.

[0321] As used herein, the terms “administration” and “administering” refer to the act of giving a drug, prodrug, or other agent, or therapeutic treatment (e.g., peptide) to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs. Exemplary routes of administration to the human body can be through space under the arachnoid membrane of the brain or spinal cord (intrathecal), the eyes (ophthalmic), mouth (oral), skin (topical or transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal or lingual), ear, rectal, vaginal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like.

[0322] As used herein, the term “treatment” or “treating” means an approach to obtaining a beneficial or intended clinical result. The beneficial or intended clinical result can include a therapeutic benefit and/or a prophylactic benefit, alleviation of symptoms, a reduction in the severity of the disease, inhibiting an underlying cause of a disease or condition, steadying diseases in a non-advanced state, delaying the progress of a disease, and/or improvement or alleviation of disease conditions. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement can be observed in the subject, notwithstanding that the subject can still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of one or more symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease can undergo treatment, even though a diagnosis of this disease may not have been made.

[0323] As used herein, the term “pharmaceutical composition” refers to the combination of an active ingredient with a carrier, inert or active, making the composition especially suitable for therapeutic or diagnostic use in vitro, in vivo or ex vivo.

[0324] The terms “pharmaceutically acceptable” or “pharmacologically acceptable,” as used herein, refer to compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject.

[0325] As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers including, but not limited to, phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), glycerol, liquid polyethylene glycols, aprotic solvents such as dimethylsulfoxide, N-methylpyrrolidone and mixtures thereof, and various types of wetting agents, solubilizing agents, anti-oxidants, bulking agents, protein carriers such as albumins, any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintegrants (e.g., potato starch or sodium starch glycolate), and the like. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, e.g., Martin, Remington's Pharmaceutical Sciences, 21st Ed., MackPubl. Co., Easton, Pa. (2005), incorporated herein by reference in its entirety.

[0326] Throughout this application, various embodiments are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0327] As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0328] As used herein, the terms “about” and “approximately,” in reference to a number, is used herein to include numbers that fall within a range of 10%, 5%, or 1% in either direction (greater than or less than) the number unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

Numbered Embodiments

[0329] The following embodiments recite non-limiting permutations of combinations of features disclosed herein. Other permutations of combinations of features are also contemplated. In particular, each of these numbered embodiments is contemplated as depending from or relating to every previous or subsequent numbered embodiment, independent of their order as listed. 1. A recombinant polynucleotide comprising: an RSV promoter; a coding sequence operably linked to the RSV promoter, wherein the coding sequence encodes a progranulin; a post- transcriptional regulatory element; a polyadenylation signal; and a transcriptional pause site. 2. The recombinant polynucleotide of embodiment 1 , wherein the post-transcriptional regulatory element comprises a WPRE. 3. The recombinant polynucleotide of embodiment 2, wherein the WPRE is a shortened WPRE. 4. The recombinant polynucleotide of embodiment 3, wherein the shortened WPRE is a WPRE3 comprising at least at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 8. 5. The recombinant polynucleotide of embodiment 3 or embodiment 4, wherein the WPRE3 comprises at least 90% sequence identity to SEQ ID NO: 8. 6. The recombinant polynucleotide of any one of embodiments 3-5, wherein the WPRE3 comprises a sequence of SEQ ID NO: 8. 7. The recombinant polynucleotide of embodiment 2, wherein the WPRE comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 7. 8. The recombinant polynucleotide of embodiment 7, wherein the WPRE comprises at least 90% sequence identity to SEQ ID NO: 7. 9. The recombinant polynucleotide of embodiment 7 or embodiment 8, wherein the WPRE comprises a sequence of SEQ ID NO: 7. 10. The recombinant polynucleotide of any one of embodiments 1-9, wherein the post- transcriptional regulatory element is downstream of the coding sequence. 11. The recombinant polynucleotide of any one of embodiments 1-10, wherein the polyadenylation signal comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 42, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 43 - SEQ ID NO: 48, or SEQ ID NO: 55. 12. The recombinant polynucleotide of any one of embodiments 1-11, wherein the polyadenylation signal comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 42. 13. The recombinant polynucleotide of any one of embodiments 1-12, wherein the polyadenylation signal comprises at least 90% sequence identity to SEQ ID NO: 42. 14. The recombinant polynucleotide of any one of embodiments 1-12, wherein the polyadenylation signal comprises a sequence of SEQ ID NO: 42. 15. The recombinant polynucleotide of any one of embodiments 1-14, wherein the polyadenylation signal is downstream of the coding sequence. 16. The recombinant polynucleotide of any one of embodiments 1-15, wherein the transcriptional pause site comprises a second polyadenylation signal. 17. The recombinant polynucleotide of any one of embodiments 1-16, wherein the transcriptional pause site comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 64. 18. The recombinant polynucleotide of any one of embodiments 1-17, wherein the transcriptional pause site comprises at least 90% sequence identity to SEQ ID NO: 64. 19. The recombinant polynucleotide of any one of embodiments 1- 18, wherein the transcriptional pause site comprises a sequence of SEQ ID NO: 64. 20. The recombinant polynucleotide of any one of embodiments 1-19, wherein the transcriptional pause site is downstream of the coding sequence. 21. The recombinant polynucleotide of any one of embodiments 1-20, wherein the transcriptional pause site is downstream of the polyadenylation signal. 22. The recombinant polynucleotide of any one of embodiments 1-21, wherein the coding sequence is downstream of the RSV promoter, the post-transcriptional regulatory element is downstream of the coding sequence, the polyadenylation signal is downstream of the post-transcriptional regulatory element, and the transcriptional pause site is downstream of the post-transcriptional regulatory element. 23. The recombinant polynucleotide of any one of embodiments 1-22, wherein the coding sequence is downstream of the RSV promoter, the post- transcriptional regulatory element is downstream of the coding sequence, the polyadenylation signal is downstream of the post-transcriptional regulatory element, and the transcriptional pause site is downstream of the polyadenylation signal. 24. The recombinant polynucleotide of any one of embodiments 1-23, wherein the RSV promoter comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 3. 25. The recombinant polynucleotide of any one of embodiments 1-24, wherein the RSV promoter comprises a sequence of SEQ ID NO: 3. 26. The recombinant polynucleotide of any one of embodiments 1-25, wherein the progranulin is a human progranulin. 27. The recombinant polynucleotide of any one of embodiments 1-26, wherein the progranulin coding sequence codes for an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 63. 28. The recombinant polynucleotide of any one of embodiments 1-27, wherein the progranulin coding sequence codes for an amino acid sequence comprising the sequence of SEQ ID NO: 63. 29. The recombinant polynucleotide of any one of embodiments 1-28, wherein the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 10. 30. The recombinant polynucleotide of any one of embodiments 1- 28, wherein the coding sequence comprises a sequence of SEQ ID NO: 10. 31. The recombinant polynucleotide of any one of embodiments 1-28, wherein the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 6. 32. The recombinant polynucleotide of any one of embodiments 1-28, wherein the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 11. 33. The recombinant polynucleotide of any one of embodiments 1-28, wherein the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 26. 34. The recombinant polynucleotide of any one of embodiments 1- 28, wherein the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 57 and at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 58. 35. The recombinant polynucleotide of any one of embodiments 1-34, further comprising a stuffer sequence. 36. The recombinant polynucleotide of embodiment 35, wherein the stuffer sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 61. 37. The recombinant polynucleotide of embodiment 35 or 36, wherein the stuffer sequence comprises a sequence of SEQ ID NO: 61. 38. The recombinant polynucleotide of any one of embodiments 35-37, wherein the stuffer sequence is upstream of the coding sequence. 39. The recombinant polynucleotide of any one of embodiments 35-38, wherein the stuffer sequence is upstream of the RSV promoter. 40. The recombinant polynucleotide of any one of embodiments 35-39, wherein the stuffer sequence is upstream of the RSV promoter, the coding sequence is downstream of the RSV promoter, the post-transcriptional regulatory element is downstream of the coding sequence, the polyadenylation signal is downstream of the post-transcriptional regulatory element, and the transcriptional pause site is downstream of the polyadenylation signal. 41. The recombinant polynucleotide of any one of embodiments 1-40, further comprising a 5’ untranslated region. 42. The recombinant polynucleotide of embodiment 41, wherein the 5’ untranslated region comprises a Kozak sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 66. 43. The recombinant polynucleotide of embodiment 41 or embodiment 42, wherein the 5’ untranslated region comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 56. 44. The recombinant polynucleotide of any one of embodiments 41-43, wherein the 5’ untranslated region comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 62. 45. The recombinant polynucleotide of any one of embodiments 41-44, wherein the 5’ untranslated region comprises a sequence of SEQ ID NO: 62. 46. The recombinant polynucleotide of any one of embodiments 41-45, wherein the 5’ untranslated region is upstream of the coding sequence. 47. The recombinant polynucleotide of any one of embodiments 41-46, wherein the 5’ untranslated region is downstream of the RSV promoter. 48. The recombinant polynucleotide of any one of embodiments 41-47, wherein the stuffer sequence is upstream of the RSV promoter, the 5 ’ untranslated region is downstream of the RSV promoter, the coding sequence is downstream of the 5 ’ untranslated region, the post- transcriptional regulatory element is downstream of the coding sequence, the polyadenylation signal is downstream of the post-transcriptional regulatory element, and the transcriptional pause site is downstream of the polyadenylation signal. 49. The recombinant polynucleotide of any one of embodiments 1-48, further comprising a 5’ inverted terminal repeat upstream of the RSV promoter and a 3’ inverted terminal repeat downstream of the transcriptional pause site. 50. The recombinant polynucleotide of any one of embodiments 1-48, further comprising a 5’ inverted terminal repeat upstream of the stuffer sequence and a 3 ’ inverted terminal repeat downstream of the transcriptional pause site. 51. The recombinant polynucleotide of any one of embodiments 1- 48, further comprising a 5’ inverted terminal repeat and a 3’ inverted terminal repeat, wherein the 5’ inverted terminal repeat is upstream of the stuffer sequence, the stuffer sequence is upstream of the RSV promoter, the 5’ untranslated region is downstream of the RSV promoter, the coding sequence is downstream of the 5 ’ untranslated region, the post-transcriptional regulatory element is downstream of the coding sequence, the polyadenylation signal is downstream of the post-transcriptional regulatory element, the transcriptional pause site is downstream of the polyadenylation signal, and the 3 ’ inverted terminal repeat downstream of the transcriptional pause site. 52. The recombinant polynucleotide of any one of embodiments 49- 51, wherein the 5’ inverted terminal repeat is an AAV 5’ inverted terminal repeat. 53. The recombinant polynucleotide of embodiment 52, wherein the AAV 5’ inverted terminal repeat is a single stranded AAV 5’ inverted terminal repeat. 54. The recombinant polynucleotide of any one of embodiments 49-53, wherein the 5’ inverted terminal repeat is an AAV5 5’ inverted terminal repeat, an AAV1 5’ inverted terminal repeat, an AAV2 5’ inverted terminal repeat, an AAV9 5’ inverted terminal repeat, or a PhP.eB 5’ inverted terminal repeat. 55. The recombinant polynucleotide of embodiment 49-54, wherein the 5’ inverted terminal repeat is an AAV2 5’ inverted terminal repeat. 56. The recombinant polynucleotide of embodiment 49-55, wherein the 5’ inverted terminal repeat is a single stranded AAV2 5’ inverted terminal repeat. 57. The recombinant polynucleotide of any one of embodiments 49-56, wherein the 5’ inverted terminal repeat comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 59. 58. The recombinant polynucleotide of any one of embodiments 49-57, wherein the 5’ inverted terminal repeat comprises a sequence of SEQ ID NO: 59. 59. The recombinant polynucleotide of any one of embodiments 49-58, wherein the 3’ inverted terminal repeat is an AAV 3’ inverted terminal repeat. 60. The recombinant polynucleotide of embodiment 59, wherein the AAV 3’ inverted terminal repeat is a single stranded AAV 3’ inverted terminal repeat. 61. The recombinant polynucleotide of any one of embodiments 49-60, wherein the 3’ inverted terminal repeat is an AAV5 3’ inverted terminal repeat, an AAV1 3’ inverted terminal repeat, an AAV2 3’ inverted terminal repeat, an AAV9 3’ inverted terminal repeat, or a PhP.eB 3’ inverted terminal repeat. 62. The recombinant polynucleotide of embodiment 49-61, wherein the 5’ inverted terminal repeat is an AAV2 3’ inverted terminal repeat. 63. The recombinant polynucleotide of embodiment 49-62, wherein the 5’ inverted terminal repeat is a single stranded AAV2 3’ inverted terminal repeat. 64. The recombinant polynucleotide of any one of embodiments 49-63, wherein the 3’ inverted terminal repeat comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 60. 65. The recombinant polynucleotide of any one of embodiments 1-64, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 3; (ii) SEQ ID NO 10; (iii) SEQ ID NO: 8; (iv) SEQ ID NO: 42; and (v) SEQ ID NO: 64. 66. The recombinant polynucleotide of any one of embodiments 1-65, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 59; (ii) SEQ ID NO: 3; (iii) SEQ ID NO 10; (iv) SEQ ID NO: 8; (v) SEQ ID NO: 42; (vi) SEQ ID NO: 64; and (vii) SEQ ID NO: 60. 67. The recombinant polynucleotide of any one of embodiments 1-66, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 3; (ii) SEQ ID NO: 62, wherein the 3’ 33 nucleotides of SEQ ID NO: 3 overlap with the 5’ 33 nucleotides of SEQ ID NO: 62; (iii) SEQ ID NO 10; (iv) SEQ ID NO: 8; (v) SEQ ID NO: 42; and (vi) SEQ ID NO: 64. 68. The recombinant polynucleotide of any one of embodiments 1-67, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 3; (ii) SEQ ID NO: 66; (iii) SEQ ID NO 10; (iv) SEQ ID NO: 8; (v) SEQ ID NO: 42; and (vi) SEQ ID NO: 64. 69. The recombinant polynucleotide of any one of embodiments 1-68, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 3; (ii) SEQ ID NO: 56; (iii) SEQ ID NO 10; (iv) SEQ ID NO: 8; (v) SEQ ID NO: 42; and (vi) SEQ ID NO: 64. 70. The recombinant polynucleotide of any one of embodiments 1-69, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 61 (ii) SEQ ID NO: 3; (iii) SEQ ID NO: 62, wherein the 3’ 33 nucleotides of SEQ ID NO: 3 overlap with the 5’ 33 nucleotides of SEQ ID NO: 62; (iv) SEQ ID NO 10; (v) SEQ ID NO: 8; (vi) SEQ ID NO: 42; and (vii) SEQ ID NO: 64. 71. The recombinant polynucleotide of any one of embodiments 1-70, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 61 (ii) SEQ ID NO: 3; (iii) SEQ ID NO: 66; (iv) SEQ ID NO 10; (v) SEQ ID NO: 8; (vi) SEQ ID NO: 42; and (vii) SEQ ID NO: 64.

72. The recombinant polynucleotide of any one of embodiments 1-71, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 61 (ii) SEQ ID NO: 3; (iii) SEQ ID NO: 56; (iv) SEQ ID NO 10; (v) SEQ ID NO: 8; (vi) SEQ ID NO: 42; and (vii) SEQ ID NO: 64.

73. The recombinant polynucleotide of any one of embodiments 1-72, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 59; (ii) SEQ ID NO: 3; (iii) SEQ ID NO: 62, wherein the 3’ 33 nucleotides of SEQ ID NO: 3 overlap with the 5’ 33 nucleotides of SEQ ID NO: 62; (iv) SEQ ID NO 10; (v) SEQ ID NO: 8; (vi) SEQ ID NO: 42; (vii) SEQ ID NO: 64; and (viii) SEQ ID NO: 60. 74. The recombinant polynucleotide of any one of embodiments 1-73, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 59; (ii) SEQ ID NO: 3; (iii) SEQ ID NO: 66; (iv) SEQ ID NO 10; (v) SEQ ID NO: 8;

(vi) SEQ ID NO: 42; (vii) SEQ ID NO: 64; and (viii) SEQ ID NO: 60. 75. The recombinant polynucleotide of any one of embodiments 1-74, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 59; (ii) SEQ ID NO: 3; (iii) SEQ ID NO: 56; (iv) SEQ ID NO 10; (v) SEQ ID NO: 8; (vi) SEQ ID NO: 42; (vii) SEQ ID NO: 64; and (viii) SEQ ID NO: 60. 76. The recombinant polynucleotide of any one of embodiments 1-75, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 59; (ii) SEQ ID NO: 61; (iii) SEQ ID NO: 3; (iv) SEQ ID NO: 62, wherein the 3’ 33 nucleotides of SEQ ID NO: 3 overlap with the 5’ 33 nucleotides of SEQ ID NO: 62; (v) SEQ ID NO 10; (vi) SEQ ID NO: 8;

(vii) SEQ ID NO: 42; (viii) SEQ ID NO: 64; and (ix) SEQ ID NO: 60. 77. The recombinant polynucleotide of any one of embodiments 1-76, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 59; (ii) SEQ ID NO: 61; (iii) SEQ ID NO: 3; (iv) SEQ ID NO: 66; (v) SEQ ID NO 10; (vi) SEQ ID NO: 8; (vii) SEQ ID NO: 42; (viii) SEQ ID NO: 64; and (ix) SEQ ID NO: 60. 78. The recombinant polynucleotide of any one of embodiments 1-77, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 59; (ii) SEQ ID NO: 61; (iii) SEQ ID NO: 3; (iv) SEQ ID NO: 56; (v) SEQ ID NO 10; (vi) SEQ ID NO: 8; (vii) SEQ ID NO: 42; (viii) SEQ ID NO: 64; and (ix) SEQ ID NO: 60. 79. The recombinant polynucleotide of any one of embodiments 1-78, wherein the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 65. 80. The recombinant polynucleotide of any one of embodiments 1-79, wherein the recombinant polynucleotide comprises a sequence of SEQ ID NO: 65. 81. The recombinant polynucleotide of any one of embodiments 1-80, wherein the recombinant polynucleotide is SEQ ID NO: 65. 82. The recombinant polynucleotide of any one of embodiments 1-81, wherein the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 121. 83. The recombinant polynucleotide of any one of embodiments 1-81, wherein the recombinant polynucleotide comprises a sequence of SEQ ID NO: 121. 84. The recombinant polynucleotide of any one of embodiments 1-81, wherein the recombinant polynucleotide is SEQ ID NO: 121. 85. The recombinant polynucleotide of any one of embodiments 1-81, wherein the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 122. 86. The recombinant polynucleotide of any one of embodiments 1-81, wherein the recombinant polynucleotide comprises a sequence of SEQ ID NO: 122. 87. The recombinant polynucleotide of any one of embodiments 1-81, wherein the recombinant polynucleotide is SEQ ID NO: 122. 88. The recombinant polynucleotide of any one of embodiments 1-81, wherein the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 123. 89. The recombinant polynucleotide of any one of embodiments 1-81, wherein the recombinant polynucleotide comprises a sequence of SEQ ID NO: 123. 90. The recombinant polynucleotide of any one of embodiments 1-81, wherein the recombinant polynucleotide is SEQ ID NO: 123. 91. The recombinant polynucleotide of any one of embodiments 1-81, wherein the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 124. 92. The recombinant polynucleotide of any one of embodiments 1-81, wherein the recombinant polynucleotide comprises a sequence of SEQ ID NO: 124. 93. The recombinant polynucleotide of any one of embodiments 1-81, wherein the recombinant polynucleotide is SEQ ID NO: 124. 94. A recombinant polynucleotide comprising: an RSV promoter; a coding sequence operably linked to the RSV promoter, wherein the coding sequence encodes a progranulin; and a post-transcriptional regulatory element. 95. The recombinant polynucleotide of embodiment 94, wherein the post-transcriptional regulatory element comprises a WPRE. 96. The recombinant polynucleotide of embodiment 95, wherein the WPRE is a shortened WPRE. 97. The recombinant polynucleotide of embodiment 96, wherein the shortened WPRE is a WPRE3 comprising at least at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 8. 98. The recombinant polynucleotide of embodiment 96 or embodiment 97, wherein the WPRE3 comprises at least 90% sequence identity to SEQ ID NO: 8. 99. The recombinant polynucleotide of any one of embodiments 98-98, wherein the WPRE3 comprises a sequence of SEQ ID NO: 8. 100. The recombinant polynucleotide of embodiment 95, wherein the WPRE comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 7. 101. The recombinant polynucleotide of embodiment 100, wherein the WPRE comprises at least 90% sequence identity to SEQ ID NO: 7. 102. The recombinant polynucleotide of embodiment 100 or embodiment 101, wherein the WPRE comprises a sequence of SEQ ID NO: 7. 103. The recombinant polynucleotide of any one of embodiments 94-102, wherein the post-transcriptional regulatory element is downstream of the coding sequence. 104. A recombinant polynucleotide comprising: an RSV promoter; a coding sequence operably linked to the RSV promoter, wherein the coding sequence encodes a progranulin; and a polyadenylation signal. 105. The recombinant polynucleotide of embodiment 104, wherein the polyadenylation signal comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 42, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 43 - SEQ ID NO: 48, or SEQ ID NO: 55. 106. The recombinant polynucleotide of embodiment 104 or embodiment 105, wherein the polyadenylation signal comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 42. 107. The recombinant polynucleotide of any one of embodiments 104-106, wherein the polyadenylation signal comprises at least 90% sequence identity to SEQ ID NO: 42. 108. The recombinant polynucleotide of any one of embodiments 104-107, wherein the polyadenylation signal comprises a sequence of SEQ ID NO: 42. 109. The recombinant polynucleotide of any one of embodiments 104-108, wherein the polyadenylation signal is downstream of the coding sequence. 110. A recombinant polynucleotide comprising: an RSV promoter; a coding sequence operably linked to the RSV promoter, wherein the coding sequence encodes a progranulin; and a transcriptional pause site. 111. The recombinant polynucleotide of embodiment 110, wherein the transcriptional pause site comprises a second polyadenylation signal. 112. The recombinant polynucleotide of embodiment 110 or embodiment 111, wherein the transcriptional pause site comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 64. 113. The recombinant polynucleotide of any one of embodiments 110-112, wherein the transcriptional pause site comprises at least 90% sequence identity to SEQ ID NO: 64. 114. The recombinant polynucleotide of any one of embodiments 110-113, wherein the transcriptional pause site comprises a sequence of SEQ ID NO: 64. 115. The recombinant polynucleotide of any one of embodiments 110-114, wherein the transcriptional pause site is downstream of the coding sequence. 116. The recombinant polynucleotide of any one of embodiments 94-115, wherein the coding sequence is downstream of the RSV promoter, the post-transcriptional regulatory element is downstream of the coding sequence, the polyadenylation signal is downstream of the post-transcriptional regulatory element, and the transcriptional pause site is downstream of the post- transcriptional regulatory element. 117. The recombinant polynucleotide of any one of embodiments 94-116, wherein the RSV promoter comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 3. 118. The recombinant polynucleotide of any one of embodiments 94-117, wherein the progranulin is a human progranulin. 119. The recombinant polynucleotide of any one of embodiments 94-118, wherein the progranulin encoded by the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 63. 120. The recombinant polynucleotide of any one of embodiments 94-119, wherein the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 6. 121. The recombinant polynucleotide of any one of embodiments 94-119, wherein the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 10. 122. The recombinant polynucleotide of any one of embodiments 94-119, wherein the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 11. 123. The recombinant polynucleotide of any one of embodiments 94-119, wherein the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 26. 124. The recombinant polynucleotide of any one of embodiments 94-119, wherein the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 57 and at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 58. 125. The recombinant polynucleotide of any one of embodiments 94-119, further comprising a stuffer sequence. 126. The recombinant polynucleotide of embodiment 125, wherein the stuffer sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 61. 127. The recombinant polynucleotide of embodiment 125 or embodiment 126, wherein the stuffer sequence is upstream of the coding sequence. 128. The recombinant polynucleotide of any one of embodiments 125-127, wherein the stuffer sequence is upstream of the RSV promoter. 129. The recombinant polynucleotide of any one of embodiments 94-128, further comprising a 5’ untranslated region. 130. The recombinant polynucleotide of embodiment 129, wherein the 5’ untranslated region comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 62, SEQ ID NO: 56, or SEQ ID NO: 66. 131. The recombinant polynucleotide of embodiment 129 or embodiment 130, wherein the 5’ untranslated region is upstream of the coding sequence. 132. The recombinant polynucleotide of any one of embodiments 129-131, wherein the 5’ untranslated region is downstream of the RSV promoter. 133. The recombinant polynucleotide of any one of embodiments 94-132, comprising a 5’ inverted terminal repeat upstream of the RSV promoter and a 3’ inverted terminal repeat downstream of the transcriptional pause site. 134. The recombinant polynucleotide of embodiment 133, wherein the 5’ inverted terminal repeat is an AAV 5’ inverted terminal repeat. 135. The recombinant polynucleotide of embodiment 133 or embodiment 134, wherein the 5’ inverted terminal repeat is an AAV5 5’ inverted terminal repeat, an AAV1 5’ inverted terminal repeat, an AAV2 5’ inverted terminal repeat, an AAV9 5’ inverted terminal repeat, or a PhP.eB 5’ inverted terminal repeat. 136. The recombinant polynucleotide of any one of embodiments 133-135, wherein the 5’ inverted terminal repeat comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 59. 137. The recombinant polynucleotide of any one of embodiments 133-136, wherein the 3’ inverted terminal repeat is an AAV 5’ inverted terminal repeat. 138. The recombinant polynucleotide of any one of embodiments 133-137, wherein the 3’ inverted terminal repeat is an AAV5 3’ inverted terminal repeat, an AAV1 3’ inverted terminal repeat, an AAV2 3’ inverted terminal repeat, an AAV9 3’ inverted terminal repeat, or a PhP.eB 3’ inverted terminal repeat. 139. The recombinant polynucleotide of any one of embodiments 133-138, wherein the 3’ inverted terminal repeat comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 60. 140. The recombinant polynucleotide of any one of embodiments 94-139, wherein the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 65. 141. The recombinant polynucleotide of any one of embodiments 1-140, further comprising a neuron-restrictive silencer element. 142. The recombinant polynucleotide of embodiment 141, wherein the neuron- restrictive silencer element comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to any one of SEQ ID NO: 32 - SEQ ID NO: 39. 143. The recombinant polynucleotide of any one of embodiments 1-142, further comprising a CCCTC-binding factor sequence. 144. The recombinant polynucleotide of embodiment 143, wherein the CCCTC-binding factor sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 38. 145. The recombinant polynucleotide of any one of embodiments 1-144, further comprising an intron. 146. The recombinant polynucleotide of embodiment 145, wherein the intron is within a 5’ untranslated region of the recombinant polynucleotide. 147. The recombinant polynucleotide of embodiment 145 or embodiment 146, wherein the intron is within the coding sequence. 148. The recombinant polynucleotide of any one of embodiments 145-147, wherein the intron comprises a GRN intron, an SV40 intron, a TPI intron, or a combination thereof. 149. The recombinant polynucleotide of embodiment 148, wherein the GRN intron comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 12. 150. The recombinant polynucleotide of embodiment 148 or embodiment 149, wherein the GRN intron comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity any one of SEQ ID NO: 27 - SEQ ID NO: 29. 151. The recombinant polynucleotide of any one of embodiments 148-150, wherein the SV40 intron comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 13. 152. The recombinant polynucleotide of any one of embodiments 148-151, wherein the SV40 intron comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 30. 153. The recombinant polynucleotide of any one of embodiments 148-152, wherein the TPI intron comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 31. 154. The recombinant polynucleotide of any one of embodiments 94-153, wherein the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 67. 155. The recombinant polynucleotide of any one of embodiments 94-153, wherein the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 68. 156. The recombinant polynucleotide of any one of embodiments 94-153, wherein the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 69. 157. The recombinant polynucleotide of any one of embodiments 94-153, wherein the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 70. 158. The recombinant polynucleotide of any one of embodiments 94-153, wherein the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 15. 159. The recombinant polynucleotide of any one of embodiments 94-153, wherein the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 16. 160. The recombinant polynucleotide of any one of embodiments 94-153, wherein the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 17. 161. The recombinant polynucleotide of any one of embodiments 94-153, wherein the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 18. 162. A plasmid comprising the recombinant polynucleotide of any one of embodiments 1-161. 163. The plasmid of embodiment 162, comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 120. 164. The plasmid of embodiment 162 or embodiment 163, wherein the plasmid is SEQ ID NO: 120. 165. The plasmid of embodiment 162, comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 19. 166. The plasmid of embodiment 162, comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 20. 167. The plasmid of embodiment 162, comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 50. 168. The plasmid of embodiment 162, comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 53. 169. The plasmid of embodiment 162, comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 71. 170. The plasmid of embodiment 162, comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 73. 171. The plasmid of embodiment 162, comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 74. 172. A viral vector comprising the recombinant polynucleotide of any one of embodiments 1-113 or the plasmid of any one of embodiments 162-171. 173. The viral vector of embodiment 117223, wherein the viral vector is an adenoviral vector, an adeno-associated viral (AAV) vector, or a lentivector. 174. The viral vector of embodiment 173, wherein the adeno-associated viral vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-DJ/8, AAV-DJ/9, AAV1/2, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAV.OligoOOl, AAV.SCH9, AAV.r3.45, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PhP.eB, AAV.PhP.Vl, AAV.PHP.B, AAV.PhB.Cl, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, AAVhu68, chimeras thereof, and combinations thereof. 175. The viral vector of embodiment 173 or embodiment 174, wherein the AAV vector is an AAV5 vector. 176. The viral vector of any one of embodiments 173-175, wherein the AAV vector comprises an engineered viral protein (VP) capsid polypeptide. 177. The viral vector of embodiment 176, wherein the engineered VP capsid polypeptide comprises at least one substitution in a 581-589 region of the engineered VP capsid polypeptide relative to a wild type VP capsid polypeptide of SEQ ID NO: 75. 178. The viral vector of embodiment 177, wherein the 581-589 region comprises a sequence of SEQ ID NO: 83. 179. The viral vector of embodiment 177, wherein the 581-589 region comprises a sequence of SEQ ID NO: 84. 180. The viral vector of embodiment 177, wherein the 581-589 region comprises a sequence of SEQ ID NO: 82. 181. The viral vector of embodiment 177, wherein the 581-589 region comprises a sequence of SEQ ID NO: 96. 182. The viral vector of embodiment 177, wherein the 581-589 region comprises a sequence of any one of SEQ ID NO: 84 - SEQ ID NO: 95 or SEQ ID NO: 97 - SEQ ID NO: 111. 183. The viral vector of any one of embodiments 177-182, wherein the engineered VP capsid polypeptide comprises a formula of (A)-(X)-(B), wherein (A) is a first polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 80, (X) is the 581-589 region, and (B) is a second polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 81. 184. A pharmaceutical composition comprising the recombinant polynucleotide of any one of embodiments 1-161, the plasmid of any one of embodiments 162-171, or the viral vector of any one of embodiments 172-183, and a pharmaceutically acceptable excipient, carrier, diluent, or combination thereof. 185. A method of expressing a progranulin in a cell, tissue, or subject, the method comprising: delivering a recombinant polynucleotide comprising an RSV promoter and a coding sequence operably linked to the RSV promoter, to the cell, tissue, or subject; and expressing a progranulin encoded by the coding sequence. 186. A method of expressing a progranulin in a cell, tissue, or subject, the method comprising: delivering the recombinant polynucleotide of any one of embodiments 1-161, the plasmid of any one of embodiments 162-171, the viral vector of any one of embodiments 172-183, or the pharmaceutical composition of embodiment 184 to the cell, tissue, or subject. 187. The method of embodiment 185 or embodiment 186, wherein the progranulin is human progranulin. 188. The method of any one of embodiments 185-187, wherein the progranulin encoded by the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 63. 189. The method of any one of embodiments 185-188, wherein the progranulin is encoded by a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 10. 190. The method of any one of embodiments 185-189, wherein the progranulin is encoded by a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 6. 191. The method of any one of embodiments 185-189, wherein the progranulin is encoded by a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 11. 192. The method of any one of embodiments 185-189, wherein the progranulin is encoded by a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 26. 193. The method of any one of embodiments 185- 189, wherein the progranulin is encoded by a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 57 and at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 58. 194. The method of any one of embodiments 185-193, comprising modulating expression of the progranulin in the cell, tissue, or subject with a neuron- restrictive silencer element. 195. The method of any one of embodiments 185-194, comprising expressing the progranulin in a fluid secreted by the cell, tissue, or subject. 196. The method of embodiment 195, wherein the progranulin is expressed in the fluid at a level of not less than 1 ng/mL and not more than 100 ng/mL. 197. The method of embodiment 195, wherein the progranulin is expressed in the fluid at a level of not less than 10 ng/mL and not more than 100 ng/mL. 198. The method of embodiment 195, wherein the progranulin is expressed in the fluid at a level of not less than 1 ng/mL and not more than 30 ng/mL. 199. The method of embodiment 195, wherein the progranulin is expressed in the fluid at a level of not less than 10 ng/mL and not more than 50 ng/mL. 200. The method of embodiment 195, wherein the progranulin is expressed in the fluid at a level of not less than 10 ng/mL and not more than 30 ng/mL. 201. The method of embodiment 195, wherein the progranulin is expressed in the fluid at a level of not less than 70 ng/mL and not more than 180 ng/mL. 202. The method of embodiment 195, wherein the progranulin is expressed in the fluid at a level of not less than 70 ng/mL and not more than 300 ng/mL. 203. The method of any one of embodiments 195-202, wherein the fluid comprises a cerebrospinal fluid. 204. A method of treating a disease or disorder in a subject in need thereof, the method comprising: delivering a recombinant polynucleotide comprising an RSV promoter and a coding sequence operably linked to the RSV promoter to the subject having frontotemporal dementia; and expressing a progranulin encoded by the coding sequence, thereby treating the subject. 205. A method of treating a disease or disorder in a subject in need thereof, the method comprising: delivering the recombinant polynucleotide of any one of embodiments 1-161, the plasmid of any one of embodiments 162- 171, the viral vector of any one of embodiments 172-183, or the pharmaceutical composition of embodiment 184 to the subject in need thereof, thereby treating the disease or disorder. 206. The method of embodiment 204 or embodiment 205, wherein the disease or disorder is frontotemporal dementia, amyotrophic lateral sclerosis (ALS), Alzheimer’s Disease, Parkinson’s Disease, stroke, Gaucher disease, arthritis, limbic-predominant age-related transactivation response DNA-binding protein 43 (TDP-43) encephalopathy, autism, neuronal ceroid lipofuscinosis (e.g., type 11 (CLN11)), dementia, and neurodegeneration; optionally, wherein the neuronal ceroid lipofuscinosis is type 11 ; optionally, wherein neurodegeneration is neurodegeneration associated with normal aging. 207. A method of treating frontotemporal dementia in a subject having frontotemporal dementia, the method comprising: delivering a recombinant polynucleotide comprising an RSV promoter and a coding sequence operably linked to the RSV promoter to the subject having frontotemporal dementia; and expressing a progranulin encoded by the coding sequence, thereby treating the subject. 208. A method of treating frontotemporal dementia in a subject having frontotemporal dementia, the method comprising: delivering the recombinant polynucleotide of any one of embodiments 1-161, the plasmid of any one of embodiments 162-171, the viral vector of any one of embodiments 172- 183, or the pharmaceutical composition of embodiment 184 to the subject having frontotemporal dementia, thereby treating the frontotemporal dementia. 209. The method of any one of embodiments 204-208, comprising expressing the progranulin in a cerebrospinal fluid of the subject. 210. The method of embodiment 209, wherein the progranulin is expressed in the cerebrospinal fluid at a level of not less than 10 ng/mL and not more than 100 ng/mL. 211. The method of embodiment 209, wherein the progranulin is expressed in the cerebrospinal fluid at a level of not less than 1 ng/mL and not more than 100 ng/mL. 212. The method of embodiment 209, wherein the progranulin is expressed in the cerebrospinal fluid at a level of not less than 10 ng/mL and not more than 30 ng/mL. 213. The method of embodiment 209, wherein the progranulin is expressed in the cerebrospinal fluid at a level of not less than 1 ng/mL and not more than 30 ng/mL. 214. The method of embodiment 209, wherein the progranulin is expressed in the cerebrospinal fluid at a level of not less than 10 ng/mL and not more than 50 ng/mL. 215. The method of embodiment 209, wherein the progranulin is expressed in the cerebrospinal fluid at a level of not less than 10 ng/mL and not more than 30 ng/mL. 216. The method of embodiment 209, wherein the progranulin is expressed in the cerebrospinal fluid at a level of not less than 70 ng/mL and not more than 180 ng/mL. 217. The method of embodiment 209, wherein the progranulin is expressed in the cerebrospinal fluid at a level of not less than 70 ng/mL and not more than 300 ng/mL. 218. The method of any one of embodiments 209-217, wherein an expression level of the progranulin in the cerebrospinal fluid is not more than 100- fold, not more than 50-fold, not more than 25-fold, not more than 20-fold, or not more than 10- fold an expression level of the progranulin in a serum of the subject. 219. The method of any one of embodiments 209-218, wherein an expression level of the progranulin in the cerebrospinal fluid is not less than 0.01-fold, not less than 0.05-fold, not less than 0.1-fold, not less than 0.2-fold, not less than 0.25-fold, or not less than 0.5-fold an expression level of the progranulin in a serum of the subject. 220. The method of any one of embodiments 209-219, wherein an expression level of the progranulin in a serum of the subject is not more than 100- fold, not more than 50-fold, not more than 25-fold, not more than 20-fold, or not more than 10- fold an expression level of the progranulin in the cerebrospinal fluid. 221. The method of any one of embodiments 209-220, wherein an expression level of the progranulin in a serum of the subject is not less than 0.01-fold, not less than 0.05-fold, not less than 0.1-fold, not less than 0.2- fold, not less than 0.25-fold, or not less than 0.5-fold an expression level of the progranulin in the cerebrospinal fluid. 222. The method of any one of embodiments 209-221, wherein an expression level of the progranulin in the cerebrospinal fluid higher than an expression level of the progranulin in a serum of the subject. 223. The method of any one of embodiments 209-222, wherein an expression level of the progranulin in the cerebrospinal fluid is not less than 0.1 -fold and not more than 100-fold an expression level of the progranulin in a serum of the subject. 224. The method of any one of embodiments 209-223, wherein an expression level of the progranulin in a serum of the subject is not more than 300 ng/mL. 225. The method of any one of embodiments 209-224, wherein an expression level of the progranulin in a serum of the subject is not more than 1500 ng/mL. 226. The method of any one of embodiments 185-225, wherein the subject is a mammal. 227. The method of embodiment 226, wherein the mammal is a human, a non-human primate, a rat, a rabbit, a mouse, or a guinea pig. The method of any one of embodiments 110-147, wherein the delivering comprises intravenous administration.

Additional Numbered Embodiments

[0330] The following embodiments recite non-limiting permutations of combinations of features disclosed herein. Other permutations of combinations of features are also contemplated. In particular, each of these numbered embodiments is contemplated as depending from or relating to every previous or subsequent numbered embodiment, independent of their order as listed. 1. A recombinant polynucleotide comprising a promoter and a sequence encoding a payload, wherein the promoter is operably linked to the sequence encoding the payload; wherein the promoter is selected from a group consisting of a CAG promoter, a CMV promoter, an RSV promoter, a JeT promoter, a synapsin promoter, a minimal CMV promoter, an EF- la promoter, a putative MECP2 promoter, a B-actin promoter, a MND promoter, and a ybTATA promoter; and wherein the payload is a progranulin. 2. The recombinant polynucleotide of embodiment 1, wherein the payload is a human progranulin. 3. The recombinant polynucleotide of embodiments 1 or 2, wherein the sequence encoding the payload is a GRN sequence. 4. The recombinant polynucleotide of any one of embodiments 1-3, wherein the sequence encoding the payload is a human GRN sequence. 5. The recombinant polynucleotide of any one of embodiments 1-4, wherein the sequence encoding the payload comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 6. 6. The recombinant polynucleotide of any one of embodiments 1-4, wherein the sequence encoding the payload comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 10. 7. The recombinant polynucleotide of any one of embodiments 1-4, wherein the sequence encoding the payload comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 11. 8. The recombinant polynucleotide of any one of embodiments 1-7, wherein the promoter is selected from the group consisting of a CAG promoter, an RSV promoter, a JeT promoter, and a synapsin promoter. 9. The recombinant polynucleotide of any one of embodiments 1-8, wherein the promoter is the CAG promoter. 10. The recombinant polynucleotide of any one of embodiments 1-9, wherein the CAG promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 1. 11. The recombinant polynucleotide of any one of embodiments 1-7, wherein the promoter is the CMV promoter. 12. The recombinant polynucleotide of any one of embodiments 1-7 or 11, wherein the CMV promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 2. 13. The recombinant polynucleotide of any one of embodiments 1-8, wherein the promoter is the RSV promoter. 14. The recombinant polynucleotide of any one of embodiments 1-8 or 13, wherein the RSV promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 3. 15. The recombinant polynucleotide of any one of embodiments 1-8, wherein the promoter is the JeT promoter. 16. The recombinant polynucleotide of any one of embodiments 1-8 or 15, wherein the JeT promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 4. 17. The recombinant polynucleotide of any one of embodiments 1-8, wherein the promoter is the Synapsin promoter. 18. The recombinant polynucleotide of any one of embodiments 1-8 or 17, wherein the Synapsin promoter is a human Synapsin (hSynapsin) promoter. 19. The recombinant polynucleotide of any one of embodiments 1-8, 17, or 18, wherein the synapsin promoter comprises at 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 5. 20. The recombinant polynucleotide of any one of embodiments 1-7, wherein the promoter is the minimal CMV promoter. 21. The recombinant polynucleotide of any one of embodiments 1-7 or 20, wherein the minimal CMV promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 21. 22. The recombinant polynucleotide of any one of embodiments 1-7, wherein the promoter is the EF- la promoter. 23. The recombinant polynucleotide of any one of embodiments 1-7 or 22, wherein the EF-la promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 22. 24. The recombinant polynucleotide of any one of embodiments 1-7, wherein the promoter is the putative MECP2 promoter. 25. The recombinant polynucleotide of any one of embodiments 1-7 or 24, wherein the putative MECP2 promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 23. 26. The recombinant polynucleotide of any one of embodiments 1-7, wherein the promoter is the B-actin promoter. 27. The recombinant polynucleotide of any one of embodiments 1-7 or 26, wherein the B-actin promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 24. 28. The recombinant polynucleotide of any one of embodiments 1-7, wherein the promoter is the MND promoter. 29. The recombinant polynucleotide of any one of embodiments 1-7 or 28, wherein the MND promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 21. 30. The recombinant polynucleotide of any one of embodiments 1-7, wherein the promoter is the ybTATA promoter. 31. The recombinant polynucleotide of any one of embodiments 1-7 or 30, wherein the ybTATA promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 54. 32. The recombinant polynucleotide of any one of embodiments 1-31, further comprising an additional sequence element. 33. The recombinant polynucleotide of embodiment 32, wherein the additional sequence element comprises a WPRE element, a neuron-restrictive silencer element, a WPRE3 element, a neuron-restrictive silencer element, a CCCTC-binding factor sequence, a polyadenylation signal, a 5 ’ untranslated region, a 3 ’ untranslated region, or combinations thereof. 34. The recombinant polynucleotide of embodiment 32 or embodiment 33, wherein the additional sequence element comprises the WPRE element. 35. The recombinant polynucleotide of embodiment 34, wherein the WPRE element comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 7. 36. The recombinant polynucleotide of any one of embodiments 32-35, wherein the additional sequence element comprises the WPRE3 element. 37. The recombinant polynucleotide of embodiment 36, wherein the WPRE3 element comprises at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 8. 38. The recombinant polynucleotide of any one of embodiments 32-37, wherein the additional sequence element comprises the neuron-restrictive silencer element. 39. The recombinant polynucleotide of embodiment 38, wherein the neuron- restrictive silencer element comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to any one of SEQ ID NO: 32 - SEQ ID NO: 39. 40. The recombinant polynucleotide of any one of embodiments 32-39, wherein the additional sequence element comprises the CCCTC-binding factor sequence. 41. The recombinant polynucleotide of embodiment 40, wherein the CCCTC-binding factor sequence comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 30. 42. The recombinant polynucleotide of any one of embodiments 32-

41, wherein the additional sequence element comprises the polyadenylation signal downstream of the sequence encoding the payload. 43. The recombinant polynucleotide of embodiment 42, wherein the polyadenylation signal comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to any one of SEQ ID NO: 40 - SEQ ID NO: 48. 44. The recombinant polynucleotide of embodiment 42 or embodiment 43, wherein the polyadenylation signal comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO:

42. 45. The recombinant polynucleotide of any one of embodiments 32-44, wherein the additional sequence element comprises the 5 ’ untranslated region upstream of the sequence encoding the payload. 46. The recombinant polynucleotide of any one of embodiments 32-45, wherein the additional sequence element comprises the 3 ’ untranslated region downstream of the sequence encoding the payload. 47. The recombinant polynucleotide of embodiment 46, wherein the 3’ untranslated region comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 40 or SEQ ID NO: 41. 48. The recombinant polynucleotide of any one of embodiments 1-47, further comprising an intron. 49. The recombinant polynucleotide of embodiment 48, wherein the intron is within a 5’ untranslated region of the recombinant polynucleotide. 50. The recombinant polynucleotide of embodiment 48 or embodiment 49, wherein the intron is within the sequence encoding the payload. 51. The recombinant polynucleotide of any one of embodiments 48-50, wherein the intron comprises a GRN intron, an SV40 intron, a TPI intron, or a combination thereof. 52. The recombinant polynucleotide of embodiment 51 , wherein the GRN intron comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 12. 53. The recombinant polynucleotide of embodiment 51 or embodiment 52, wherein the GRN intron comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity any one of SEQ ID NO: 27 - SEQ ID NO: 29. 54. The recombinant polynucleotide of any one of embodiments 48-53, wherein the SV40 intron comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 13. 55. The recombinant polynucleotide of any one of embodiments 48-54, wherein the SV40 intron comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 30. 56. The recombinant polynucleotide of any one of embodiments 48-55, wherein the TPI intron comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 31. 57. The recombinant polynucleotide of any one of embodiments 1-56, wherein the recombinant polynucleotide comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 14. 58. The recombinant polynucleotide of any one of embodiments 1-56, wherein the recombinant polynucleotide comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 15. 59. The recombinant polynucleotide of any one of embodiments 1-56, wherein the recombinant polynucleotide comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 16. 60. The recombinant polynucleotide of any one of embodiments 1-56, wherein the recombinant polynucleotide comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 17. 61. The recombinant polynucleotide of any one of embodiments 1-56, wherein the recombinant polynucleotide comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 18. 62. A recombinant polynucleotide comprising a promoter and a sequence encoding a payload, wherein the promoter is operably linked to the sequence encoding the payload, wherein the sequence encoding the payload comprises an intron, wherein the payload is progranulin, and wherein the intron is a GRN intron 9. 63. The recombinant polynucleotide of embodiment 62, wherein the GRN intron 9 comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 12. 64. The recombinant polynucleotide of embodiment 62 or embodiment 63, wherein the progranulin is a human progranulin. 65. The recombinant polynucleotide of any one of embodiments 62-64, wherein the sequence encoding the payload comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 11. 66. The recombinant polynucleotide of any one of embodiments 62-65, further comprising a 5’ untranslated region upstream of the sequence encoding the payload. 67. The recombinant polynucleotide of embodiment 66, further comprising a second intron within the 5’ untranslated region. 68. The recombinant polynucleotide of embodiment 67, wherein the second intron is an SV40 intron. 69. The recombinant polynucleotide of embodiment 68, wherein the SV40 intron comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 13. 70. A recombinant polynucleotide comprising a promoter, a sequence encoding a payload, and a 5 ’ untranslated region upstream of the sequence encoding the payload, wherein the promoter is operably linked to the sequence encoding the payload, wherein the payload is progranulin, wherein the 5’ untranslated region comprises an intron, and wherein the intron is an SV40 intron. 71. The recombinant polynucleotide of embodiment 70, wherein the SV40 intron comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 13. 72. The recombinant polynucleotide of embodiment 70 or embodiment 71, wherein the progranulin is a human progranulin. 73. The recombinant polynucleotide of any one of embodiments 70-72, wherein the sequence encoding the payload comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 11. 74. The recombinant polynucleotide of any one of embodiments 70-72, wherein the sequence encoding the payload comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 6. 75. The recombinant polynucleotide of any one of embodiments 70-72, wherein the sequence encoding the payload comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 10. 76. The recombinant polynucleotide of any one of embodiments 70- 75, comprising a second intron within the sequence encoding the payload. 77. The recombinant polynucleotide of embodiment 76, wherein the second intron is a GRN intron 9. 78. The recombinant polynucleotide of embodiment 77, wherein the GRN intron 9 comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 12. 79. The recombinant polynucleotide of any one of embodiments 62-78, wherein the promoter is selected from a group consisting of a CAG promoter, a CMV promoter, an RSV promoter, a JeT promoter, a synapsin promoter, a minimal CMV promoter, an EF- la promoter, a putative MECP2 promoter, a B-actin promoter, a MND promoter, and a ybTATA promoter. 80. The recombinant polynucleotide of any one of embodiments 60-79, wherein the promoter is selected from the group consisting of a CAG promoter, an RSV promoter, a JeT promoter, and a synapsin promoter. 81. The recombinant polynucleotide of embodiment 79 or embodiment 80, wherein the promoter is the CAG promoter. 82. The recombinant polynucleotide of embodiment 81, wherein the CAG promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 1. 83. The recombinant polynucleotide of embodiment 79, wherein the promoter is the CMV promoter. 84. The recombinant polynucleotide of embodiment 83, wherein the CMV promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 2. 85. The recombinant polynucleotide of embodiment 79 or embodiment 80, wherein the promoter is the RSV promoter. 86. The recombinant polynucleotide of embodiment 85, wherein the RSV promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 3. 87. The recombinant polynucleotide of embodiment 79 or embodiment 80, wherein the promoter is the JeT promoter. 88. The recombinant polynucleotide of embodiment 87, wherein the JeT promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 4. 89. The recombinant polynucleotide of embodiment 79 or embodiment 80, wherein the promoter is the Synapsin promoter. 90. The recombinant polynucleotide of embodiment 89, wherein the Synapsin promoter is a human Synapsin (hSynapsin) promoter. 91. The recombinant polynucleotide of embodiment 89 or embodiment 90, wherein the synapsin promoter comprises at 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 5. 92. The recombinant polynucleotide of embodiment 79, wherein the promoter is the minimal CMV promoter. 93. The recombinant polynucleotide of embodiment 92, wherein the minimal CMV promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 21. 94. The recombinant polynucleotide of embodiment 79, wherein the promoter is the EF- la promoter. 95. The recombinant polynucleotide of embodiment 94, wherein the EFla promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 22. 96. The recombinant polynucleotide of embodiment 79, wherein the promoter is the putative MECP2 promoter. 97. The recombinant polynucleotide of embodiment 96, wherein the putative MECP2 promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 23. 98. The recombinant polynucleotide of embodiment 79, wherein the promoter is the B-actin promoter. 99. The recombinant polynucleotide of embodiment 98, wherein the B-actin promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 24. 100. The recombinant polynucleotide of embodiment 79, wherein the promoter is the MND promoter. 101. The recombinant polynucleotide of embodiment 100, wherein the MND promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 21. 102. The recombinant polynucleotide of embodiment 79, wherein the promoter is the ybTATA promoter. 103. The recombinant polynucleotide of embodiment 102, wherein the ybTATA promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 54. 104. The recombinant polynucleotide of any one of embodiments 1-29, further comprising an additional sequence element. 105. The recombinant polynucleotide of embodiment 30, wherein the additional sequence element comprises a WPRE element, a neuron-restrictive silencer element, a WPRE3 element, a neuron-restrictive silencer element, a CCCTC-binding factor sequence, a polyadenylation signal, a 5 ’ untranslated region, a 3 ’ untranslated region, or combinations thereof. 106. The recombinant polynucleotide of embodiment 30 or embodiment 31, wherein the additional sequence element comprises the WPRE element. 107. The recombinant polynucleotide of embodiment 32, wherein the WPRE element comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 7. 108. The recombinant polynucleotide of any one of embodiments 30-33, wherein the additional sequence element comprises the WPRE3 element. 109. The recombinant polynucleotide of embodiment 34, wherein the WPRE3 element comprises at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 8. 110. The recombinant polynucleotide of any one of embodiments 30-35, wherein the additional sequence element comprises the neuron-restrictive silencer element. 111. The recombinant polynucleotide of embodiment 36, wherein the neuron- restrictive silencer element comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to any one of SEQ ID NO: 32 - SEQ ID NO: 39. 112. The recombinant polynucleotide of any one of embodiments 30-37, wherein the additional sequence element comprises the CCCTC-binding factor sequence. 113. The recombinant polynucleotide of embodiment 38, wherein the CCCTC-binding factor sequence comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 30. 114. The recombinant polynucleotide of any one of embodiments 30-39, wherein the additional sequence element comprises the polyadenylation signal downstream of the sequence encoding the payload. 115. The recombinant polynucleotide of embodiment 40, wherein the polyadenylation signal comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to any one of SEQ ID NO: 40 - SEQ ID NO: 48. 116. The recombinant polynucleotide of embodiment 40 or embodiment 41, wherein the polyadenylation signal comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 42. 117. The recombinant polynucleotide of any one of embodiments 30-42, wherein the additional sequence element comprises the 5 ’ untranslated region upstream of the sequence encoding the payload. 118. The recombinant polynucleotide of any one of embodiments 30-43, wherein the additional sequence element comprises the 3 ’ untranslated region downstream of the sequence encoding the payload. 119. The recombinant polynucleotide of embodiment 44, wherein the 3’ untranslated region comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 40 or SEQ ID NO: 41. 120. A plasmid comprising the recombinant polynucleotide of any one of embodiments 1-119. 121. The plasmid of embodiment 120, wherein the recombinant polynucleotide is flanked by inverted terminal repeats. 122. The plasmid of embodiment 120 or 121, comprising at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 9. 123. The plasmid of embodiment 120 or 121, comprising at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 19. 124. The plasmid of embodiment 120 or 121, comprising at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 20. 125. The plasmid of embodiment 120 or 121, comprising at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 49. 126. The plasmid of embodiment 120 or 121, comprising at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 50. 127. The plasmid of embodiment 120 or 121, comprising at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 51. 128. The plasmid of embodiment 120 or 121, comprising at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 52. 129. The plasmid of embodiment 120 or 121, comprising at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 53. 130. A viral vector encapsidating the recombinant polynucleotide of any one of embodiments 1-119. 131. The viral vector of embodiment 130, wherein the recombinant polynucleotide comprises at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to the sequence of the recombinant polynucleotide between inverted terminal repeats in SEQ ID NO: 9. 132. The viral vector of embodiment 130 or 131, wherein the viral vector is an adenoviral vector, an adeno- associated viral vector, or a lentivector. 133. The viral vector of embodiment 132, wherein the adeno-associated viral vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-DJ/8, AAV-DJ/9, AAV1/2, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAV.OligoOOl, AAV.SCH9, AAV.r3.45, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PhP.eB, AAV.PhP.Vl, AAV.PHP.B, AAV.PhB.Cl, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, AAVhu68, chimeras thereof, and combinations thereof. 134. A pharmaceutical composition comprising the recombinant polynucleotide of any one of embodiments 1-119, the plasmid of any one of embodiments 120-129, or the viral vector of any one of embodiments 130-133, and a pharmaceutically acceptable excipient, carrier, diluent, or combination thereof. 135. A method of expressing a payload in a cell, tissue, or subject, the method comprising: delivering the recombinant polynucleotide of any one of embodiments 1-119, the plasmid of any one of embodiments 120-129, the viral vector of any one of embodiments 130-133, or the pharmaceutical composition of embodiment 134 to the cell, tissue, or subject. 136. The method of embodiment 135, wherein the payload is a progranulin. 137. The method of embodiment 136, wherein the progranulin is human progranulin. 138. The method of embodiment 136 or embodiment 137, wherein the progranulin comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 6. 139. The method of embodiment 136 or embodiment 137, wherein the progranulin comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 10. 140. The method of embodiment 136 or embodiment 137, wherein the progranulin comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 11. 141. The method of any one of embodiments 135-140, comprising modulating expression of the payload in the cell, tissue, or subject with a tissue-specific promoter or a cell-specific promoter. 142. The method of any one of embodiments 135-141, comprising modulating expression of the payload in the cell, tissue, or subject with a neuron-restrictive silencer element. 143. A method of expressing a progranulin in a cell, tissue, or subject, the method comprising: delivering the recombinant polynucleotide of any one of embodiments 1-119, the plasmid of any one of embodiments 120-129, the viral vector of any one of embodiments 130-133, or the pharmaceutical composition of embodiment 134 to the cell, tissue, or subject. 144. The method of embodiment 143, comprising modulating expression of the progranulin in the cell, tissue, or subject with a tissue-specific promoter or a cell-specific promoter. 145. The method of embodiment 143 or embodiment 144, comprising modulating expression of the progranulin in the cell, tissue, or subject with a neuron-restrictive silencer element. 146. A method of treating a disease or disorder in a subject in need thereof, the method comprising: delivering the recombinant polynucleotide of any one of embodiments 1- 119, the plasmid of any one of embodiments 120-129, the viral vector of any one of embodiments 130-133, or the pharmaceutical composition of embodiment 134 to the subject in need thereof, thereby treating the disease or disorder. 147. The method of embodiment 146, wherein the disease or disorder is frontotemporal dementia, amyotrophic lateral sclerosis (ALS), Alzheimer’s Disease, Parkinson’s Disease, stroke, Gaucher disease, arthritis, limbic- predominant age-related transactivation response DNA-binding protein 43 (TDP-43) encephalopathy, autism, neuronal ceroid lipofuscinosis (e.g., type 11 (CLN11)), dementia, and neurodegeneration; optionally, wherein the neuronal ceroid lipofuscinosis is type 11 ; optionally, wherein neurodegeneration is neurodegeneration associated with normal aging. 148. A method of treating frontotemporal dementia in a subject having frontotemporal dementia, the method comprising: delivering the recombinant polynucleotide of any one of embodiments 1-119, the plasmid of any one of embodiments 120-129, the viral vector of any one of embodiments ISO- 133, or the pharmaceutical composition of embodiment 134 to the subject having frontotemporal dementia, thereby treating the frontotemporal dementia. 149. The method of any one of embodiments 146-148, further comprising expressing a progranulin in a cerebrospinal fluid of the subject. 150. The method of embodiment 149, wherein an expression level of the progranulin in the cerebrospinal fluid is not less than 1 ng/mL and not more than 3 ng/mL, not less than 2 ng/mL and not more than 4.5 ng/mL, or not less than 1 ng/mL and not more than 10 ng/mL. 151. The method of embodiment 149 or embodiment 150, wherein an expression level of the progranulin in the cerebrospinal fluid is not less than 1 ng/mL and not more than 4 ng/mL. 152. The method of any one of embodiments 149-151, wherein an expression level of the progranulin in the cerebrospinal fluid is not less than 3 ng/mL. 153. The method of any one of embodiments 149-152, wherein an expression level of the progranulin in the cerebrospinal fluid is not more than 100-fold, not more than 50-fold, not more than 25-fold, not more than 20-fold, or not more than 10-fold an expression level of the progranulin in a serum of the subject. 154. The method of any one of embodiments 149-153, wherein an expression level of the progranulin in the cerebrospinal fluid is not less than 0.01-fold, not less than 0.05-fold, not less than 0.1-fold, not less than 0.2-fold, not less than 0.25-fold, or not less than 0.5-fold an expression level of the progranulin in a serum of the subject. 155. The method of any one of embodiments 149-154, wherein an expression level of the progranulin in a serum of the subject is not more than 100- fold, not more than 50-fold, not more than 25-fold, not more than 20-fold, or not more than 10- fold an expression level of the progranulin in the cerebrospinal fluid. 156. The method of any one of embodiments 149-155, wherein an expression level of the progranulin in a serum of the subject is not less than 0.01-fold, not less than 0.05-fold, not less than 0.1-fold, not less than 0.2- fold, not less than 0.25-fold, or not less than 0.5-fold an expression level of the progranulin in the cerebrospinal fluid. 157. The method of any one of embodiments 149-156, wherein an expression level of the progranulin in the cerebrospinal fluid higher than an expression level of the progranulin in a serum of the subject. 158. The method of any one of embodiments 149-157, wherein an expression level of the progranulin in the cerebrospinal fluid is not less than 0.1 -fold and not more than 100-fold an expression level of the progranulin in a serum of the subject. 159. The method of any one of embodiments 135-158, wherein the subject is a mammal. 160. The method of embodiment 159, wherein the mammal is a human, a non-human primate, a rat, a rabbit, a mouse, or a guinea pig.

Further Numbered Embodiments

[0331] The following embodiments recite non-limiting permutations of combinations of features disclosed herein. Other permutations of combinations of features are also contemplated. In particular, each of these numbered embodiments is contemplated as depending from or relating to every previous or subsequent numbered embodiment, independent of their order as listed. 1. A recombinant polynucleotide comprising a promoter and a sequence encoding a payload, wherein the promoter is operably linked to the sequence encoding the payload; wherein the promoter is selected from a group consisting of a CAG promoter, a CMV promoter, a SV40 promoter, an RSV promoter, a JeT promoter, and a synapsin promoter; and wherein the payload is progranulin. 2. The recombinant polynucleotide of embodiment 1, wherein the payload is human progranulin. 3. The recombinant polynucleotide of embodiments 1 or 2, wherein a sequence of the payload is a GRN sequence; optionally, wherein the sequence of the payload is a human GRN sequence. 4. The recombinant polynucleotide of any one of embodiments 1-3, wherein the sequence encoding the payload comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 6. 5. The recombinant polynucleotide of any one of embodiments 1-4, wherein the promoter is the CAG promoter. 6. The recombinant polynucleotide of any one of embodiments 1-5, wherein the CAG promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 1. 7. The recombinant polynucleotide of any one of embodiments 1-4, wherein the promoter is the CMV promoter. 8. The recombinant polynucleotide of any one of embodiments 1-4 or 7, wherein the CMV promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 2. 9. The recombinant polynucleotide of any one of embodiments 1-4, wherein the promoter is the RSV promoter. 10. The recombinant polynucleotide of any one of embodiments 1-4 or 9, wherein the RSV promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 3. 11. The recombinant polynucleotide of any one of embodiments 1-4, wherein the promoter is the JeT promoter. 12. The recombinant polynucleotide of any one of embodiments 1-4 or 11, wherein the JeT promoter comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 4. 13. The recombinant polynucleotide of any one of embodiments 1-4, wherein the promoter is the Synapsin promoter. 14. The recombinant polynucleotide of any one of embodiments 1-4 or 13, wherein the Synapsin promoter is a human Synapsin (hSynapsin) promoter. 15. The recombinant polynucleotide of any one of embodiments 1-4, 13, or 14, wherein the synapsin promoter comprises at 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 5. 16. The recombinant polynucleotide of any one of embodiments 1-4, wherein the promoter is the SV40 promoter. 17. The recombinant polynucleotide of any one of embodiments 1-16, further comprising a WPRE element. 18. The recombinant polynucleotide of any one of embodiments 1-17, wherein the WPRE element comprises at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 7. 19. The recombinant polynucleotide of any one of embodiments 1-16, further comprising a WPRE3 element. 20. The recombinant polynucleotide of any one of embodiments 1-16 or 19, wherein the WPRE3 element comprises at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 8. 21. A plasmid comprising the recombinant polynucleotide of any one of embodiments 1-20. 22. The plasmid of embodiment 21, wherein the recombinant polynucleotide is flanked by ITRs. 23. The plasmid of embodiment 21 or 22 comprising at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 9. 24. A viral vector encapsidating the recombinant polynucleotide of any one of embodiments 1-20. 25. The viral vector of embodiment 24, wherein the recombinant polynucleotide comprises at least at least 80%, 85%, 90%, 95%, 97%, 99% or 100% sequence identity to the sequence of the recombinant polynucleotide between ITRs in SEQ ID NO: 9. 26. The viral vector of embodiment 24 or 25, wherein the viral vector is an adenoviral vector, an adeno-associated viral vector, or a lentivector. 27. The viral vector of embodiment 26, wherein the adeno-associated viral vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-DJ/8, AAV-DJ/9, AAV1/2, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAV.OligoOOl, AAV.SCH9, AAV.r3.45, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PhP.eB, AAV.PhP.Vl, AAV.PHP.B, AAV.PhB.Cl, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, AAVhu68, chimeras thereof, and combinations thereof. 28. A pharmaceutical composition comprising the recombinant polynucleotide of any one of embodiments 1-20, the plasmid of any one of embodiments 21-23, or the viral vector of any one of embodiments 24-27, and a pharmaceutically acceptable excipient, carrier, diluent, or combination thereof. 29. A method of expressing a payload in a cell, tissue, or subject, the method comprising: delivering the recombinant polynucleotide of any one of embodiments 1-20, the plasmid of any one of 21-23, the viral vector of any one of embodiments 24-27, or the pharmaceutical composition of embodiment 28 to the cell, tissue, or subject. 30. A method of expressing progranulin in a cell, tissue, or subject, the method comprising: delivering the recombinant polynucleotide of any one of embodiments 1-20, the plasmid of any one of 21-23, the viral vector of any one of embodiments 24-27, or the pharmaceutical composition of embodiment 28 to the cell, tissue, or subject. 31. A method of treating a disease or disorder in a subject in need thereof, the method comprising: delivering the recombinant polynucleotide of any one of embodiments 1-20, the plasmid of any one of embodiments 21-23, the viral vector of any one of embodiments 24-27, or the pharmaceutical composition of embodiment 28 to the subject in need thereof, thereby treating the disease or disorder. 32. A method of treating frontotemporal dementia in a subject having frontotemporal dementia, the method comprising: delivering the recombinant polynucleotide of any one of embodiments 1-20, the plasmid of any one of embodiments 21-23, the viral vector of any one of embodiments 24-27, or the pharmaceutical composition of embodiment 28 to the subject having frontotemporal dementia, thereby treating the frontotemporal dementia. 33. The method of any one of embodiments 29-32, wherein the subject is a mammal. 34. The method of 33, wherein the mammal is a human, a non-human primate, a rat, a rabbit, a mouse, or a guinea pig.

EXAMPLES

[0332] The invention is further illustrated by the following non-limiting examples.

EXAMPLE 1

Detection of Progranulin from Recombinant Polynucleotides in Vivo [0333] This example describes detection of progranulin expression from a recombinant polynucleotide comprising a CAG promoter (SEQ ID NO: 1) operably linked to a sequence encoding progranulin (SEQ ID NO: 6) and comprising a WPRE (SEQ ID NO: 7), after administration to mice.

[0334] A plasmid comprising a recombinant polynucleotide having a CAG promoter (SEQ ID NO: 1) operably linked to a sequence encoding progranulin (SEQ ID NO: 6) and having a WPRE (SEQ ID NO: 7), a chimeric intron, and a rabbit beta globin polyA (SEQ ID NO: 42), flanked by ITRs was produced (SEQ ID NO: 9), as shown in FIG. 1. This plasmid was used to produce ssAAV9-CAG-GRN virus (encapsidating the recombinant polynucleotide) via triple transfection.

[0335] The produced ssAAV9-CAG-GRN virus was then tested for its ability to infect cells and express progranulin. This was done by infecting HEK293 cells in the presence of compound C and then testing the supernatant for progranulin. As shown in FIG. 2, progranulin was detected in supernatant from HEK293 cells infected at either 6xl0⁴ vg/cell or 2xl0⁴ vg/cell compared to no progranulin detection in the cells not infected by AAV9 virus, indicating that the produced AAV9-CAG-GRN successfully infected cells and expressed progranulin.

[0336] Next, female C57BL6 mice were administered an intracranial ventricular (ICV) injection of either a control scAAV9-CMV-GFP control virus (recombinant polynucleotide comprising a CMV promoter operably linked to GFP) at 2.O4xlO¹⁰ vg/mouse or ssAAV9-CAG-GRN virus at 5xlO¹⁰ vg/mouse. At weeks 3, 4, and 6, CSF, brain tissue, and serum was collected.

[0337] The body weights of the mice were measured after ICV to assess recovery after ICV injection and tolerance of the treatments. As shown in FIG. 3, mice exhibited a small percent weight loss in the 2-3 days post ICV surgery, but upon recovery, the mice continued to gain weight over the course of the treatment (3 weeks, 4 weeks, and 6 weeks after ICV injection, from left to right; top row shows mice injected with ssAAV9-CAG-GRN and bottom row shows mice injected with the scAAV9-CMV-GFP control).

[0338] After collection of serum and CSF, Enzyme-Linked Immunosorbant Assays (ELISAs) were performed to quantify the amount of progranulin produced in the mice. Progranulin was detected in the CSF at high levels and continued to rise over time up to the 6 week time point as shown in FIG. 4. The mean CSF progranulin levels (N=8/timepoint) were as follows: 84.25 ng/mL at week 3 post ICV injection; 179.62 ng/mL at week 4 post ICV injection; and 242.13 ng/mL at week 6 post ICV injection. Progranulin expression at each time point was significantly higher, as determined using Tukey’s honest significant difference (HSD) test, than the GFP control. This progranulin was shown to be specific to human Progranulin since there was no detection of human progranulin in the control sample (ELISA did not detect murine Progranulin). Progranulin was also detected at high levels in the serum as shown in FIG. 5, which indicates that AAV9 likely crosses the blood brain barrier after ICV injection to infect cells in the periphery and subsequently express progranulin. The mean serum progranulin levels (N=8/timepoint) were 22,854 ng/mL at 2 weeks post-ICV injection; 5,695 ng/mL at 3 weeks post-ICV injection; 7,037 ng/mL at 4 weeks post-ICV injection; and 14,684 ng/mL at 6 weeks post-ICV injection. Although over-expression of progranulin has been demonstrated to be associated with immune-mediated toxicities, no overt effects on mouse health were observed. As shown in FIG. 6, there was a slight correlation of serum levels with CSF levels. Brain tissue is assessed via histology and viral genomes per diploid genomes (vg/dg) is also assessed. EXAMPLE 2

Detection of Progranulin from Recombinant Polynucleotides in Vivo

[0339] This example describes detection of progranulin expression from a recombinant polynucleotide comprising a CMV promoter (SEQ ID NO: 2) operably linked to a sequence encoding progranulin (SEQ ID NO: 6) and comprising a WPRE (SEQ ID NO: 7); a recombinant polynucleotide comprising a RSV promoter (SEQ ID NO: 3) operably linked to a sequence encoding progranulin (SEQ ID NO: 6) and comprising a WPRE (SEQ ID NO: 7); a recombinant polynucleotide comprising a JeT promoter (SEQ ID NO: 4) operably linked to a sequence encoding progranulin (SEQ ID NO: 6) and comprising a WPRE (SEQ ID NO: 7); or a recombinant polynucleotide comprising a hSynapsin promoter (SEQ ID NO: 5) operably linked to a sequence encoding progranulin (SEQ ID NO: 6) and comprising a WPRE (SEQ ID NO: 7); after administration to mice.

[0340] A plasmid comprising a recombinant polynucleotide having a CMV promoter (SEQ ID NO: 2) operably linked to a sequence encoding progranulin (SEQ ID NO: 6) and having a WPRE (SEQ ID NO: 7) flanked by ITRs was produced. This plasmid was used to produce ssAAV9-CMV-GRN virus (encapsidating the recombinant polynucleotide) via triple transfection. A plasmid comprising a recombinant polynucleotide having a RSV promoter (SEQ ID NO: 3) operably linked to a sequence encoding progranulin (SEQ ID NO: 6) and having a WPRE (SEQ ID NO: 7) flanked by ITRs was produced. This plasmid was used to produce ssAAV9-RSV-GRN virus (encapsidating the recombinant polynucleotide) via triple transfection. A plasmid comprising a recombinant polynucleotide having a JeT promoter (SEQ ID NO: 4) operably linked to a sequence encoding progranulin (SEQ ID NO: 6) and having a WPRE (SEQ ID NO: 7) flanked by ITRs was produced. This plasmid was used to produce ssAAV9-JeT-GRN virus (encapsidating the recombinant polynucleotide) via triple transfection. A plasmid comprising a recombinant polynucleotide having a hSynapsin promoter (SEQ ID NO: 5) operably linked to a sequence encoding progranulin (SEQ ID NO: 6) and having a WPRE (SEQ ID NO: 7) flanked by ITRs was produced. This plasmid was used to produce ssAAV9- hSynapsin-GRN virus (encapsidating the recombinant polynucleotide) via triple transfection.

[0341] The produced ssAAV9-CMV-GRN virus, ssAAV9-RSV-GRN virus, ssAAV9-JeT-GRN virus, and ssAAV9-hSynapsin-GRN virus were then tested for their ability to infect cells and express progranulin by infecting HEK293 cells in the presence of compound C and then testing the supernatant for progranulin. As shown in FIG. 16A and FIG. 16B, progranulin expression was measured in HEK293 cells infected with 20,000 vg/cell (FIG. 16A) or 60,000 vg/cell (FIG. 16B) of scAAV9 encapsidating a JeT promoter (SEQ ID NO: 4) operably linked to GRN coding sequence (SEQ ID NO: 6; “scAAV9 JET PGRN”), scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6; “scAAV9 RSV PGRN”), scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6) and SV40 intron (SEQ ID NO: 13; “scAAV9 RSV SV40intron99”), scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence containing GRN intron 9 (SEQ ID NO: 11; “scAAV9 RSV PGRN int9”), ssAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6; “ssAAV9 RSV PGRN”), ssAAV9 encapsidating a CAG promoter (SEQ ID NO: 1) operably linked to GRN coding sequence (SEQ ID NO: 6) and WPRE sequence (SEQ ID NO: 7; “ssAAV9 CAG PGRN WPRE”), ssAAV9 encapsidating a hSyn promoter (SEQ ID NO: 5) operably linked to GRN coding sequence (SEQ ID NO: 6) and WPRE sequence (SEQ ID NO: 7; “ssAAV9 hSyn PGRN WPRE”), or ssAAV9 encapsidating a CMV promoter (SEQ ID NO: 2) operably linked to GRN coding sequence (SEQ ID NO: 6) and WPRE sequence (SEQ ID NO: 7; “ssAAV9 CMV PGRN WPRE”).

[0342] Next, female C57BL6 mice were administered an intracranial ventricular (ICV) injection of a control scAAV9-CMV-GFP control virus (recombinant polynucleotide comprising a CMV promoter operably linked to GFP), ssAAV9-CMV-GRN virus, ssAAV9-RSV-GRN virus, ssAAV9-JeT-GRN virus, or ssAAV9-hSynapsin-GRN virus. At week 4 CSF, brain tissue, and serum were collected.

[0343] The body weights of the mice were measured after ICV to assess recovery after ICV injection and tolerance of the treatments. As shown in FIG. 7A and FIG. 7B, some mice exhibited a small percent weight loss in the 2-3 days post ICV surgery, but upon recovery, the mice continued to gain weight over the course of the treatment, indicating that the treatment was well tolerated. FIG. 7A shows body weight data after infection with virus encoding hGRN (SEQ ID NO: 6) under control of a RSV promoter (SEQ ID NO: 3, top left), hGRN (SEQ ID NO: 6) under control of a JET promoter (SEQ ID NO: 4, top right), hGRN (SEQ ID NO: 6) under control of a RSV promoter (SEQ ID NO: 3, bottom left), and hGRN (SEQ ID NO: 6) under control of a CAG promoter (SEQ ID NO: 1) with a WPRE (SEQ ID NO: 7, bottom right). FIG. 7B shows body weight data after infection with virus encoding hGRN (SEQ ID NO: 6) with an SV40 intron (SEQ ID NO: 13) under control of a RSV promoter (SEQ ID NO: 3, top left), GRN (SEQ ID NO: 6) with an intron 9 (SEQ ID NO: 12) under control of a RSV promoter (SEQ ID NO: 3, top right), hGRN (SEQ ID NO: 6) under control of a hSynapsin promoter (SEQ ID NO: 5) with a WPRE (SEQ ID NO: 7, bottom left), and hGRN (SEQ ID NO: 6) under control of a CMV promoter (SEQ ID NO: 2) with a WPRE (SEQ ID NO: 7, bottom right). [0344] After collection of serum and CSF, Enzyme-Linked Immunosorbent Assays (ELISAs) were performed to quantify the amount of progranulin produced in the mice. Mice were infected with scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6) (“scAAV9-RSV_hGRN”; AAV produced using a plasmid of SEQ ID NO: 50), scAAV9 encapsidating a JeT promoter (SEQ ID NO: 4) operably linked to GRN coding sequence (SEQ ID NO: 6) (“scAAV9-JET_hGRN”; AAV produced using a plasmid of SEQ ID NO: 49), scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6) and SV40 intron (SEQ ID NO: 13) (“scAAV9- RSV_hGRN_SV40int99”; AAV produced using a plasmid of SEQ ID NO: 19), scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence containing GRN intron 9 (SEQ ID NO: 11) (“scAAV9-RSV_hGRN_int9”; AAV produced using a plasmid of SEQ ID NO: 20), ssAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6) (“ssAAV9-RSV-hGRN”; AAV produced using a plasmid of SEQ ID NO: 53), ssAAV9 encapsidating a CAG promoter (SEQ ID NO: 1) operably linked to GRN coding sequence (SEQ ID NO: 6) and WPRE sequence (SEQ ID NO: 7) (“ssAAV9-CAG_hGRN_WPRE”; AAV produced using a plasmid of SEQ ID NO: 9), ssAAV9 encapsidating a hSyn promoter (SEQ ID NO: 5) operably linked to GRN coding sequence (SEQ ID NO: 6) and WPRE sequence (SEQ ID NO: 7) (“ssAAV9- Syn_hGRN_WPRE”; AAV produced using a plasmid of SEQ ID NO: 52), or ssAAV9 encapsidating a CMV promoter (SEQ ID NO: 2) operably linked to GRN coding sequence (SEQ ID NO: 6) and WPRE sequence (SEQ ID NO: 7) (“ssAAV9-CMV_hGRN_WPRE;” AAV produced using a plasmid of SEQ ID NO: 51). As shown in FIG. 8A and FIG. 8B, each of the AAVs produced via triple transfection of plasmids comprising the recombinant polynucleotides expressed progranulin in the CSF of the mice. AAVs were produced from plasmids encoding for each of the recombinant polynucleotides tested, including ssAAV9-Syn_hGRN_WPRE (SEQ ID NO: 52) as shown in FIG. 9, produced progranulin in CSF at levels above the target therapeutic level of 3 ng/mL (dotted line in FIG. 9).

[0345] Progranulin was also detected in the serum of mice at both 2 week (FIG. 10) and 4 week (FIG. 11) time points. Mean progranulin levels produced by each recombinant polynucleotide in serum (at 2 weeks and 4 weeks) and CSF are provided in TABLE 8. TABLE 8. Mean CSF Progranulin Levels

[0346] As shown in TABLE 8, each of the tested recombinant polynucleotides exhibited serum to CSF progranulin expression ratios of less than 100. AAV produced using a plasmid of SEQ ID NO: 52, containing a hSynapsin promoter (SEQ ID NO: 5) with a WPRE (SEQ ID NO: 7), produced a serum to CSF progranulin expression ratio of less than 1, demonstrating higher production of progranulin in CSF than in serum. AAV produced using a plasmid of SEQ ID NO: 53, containing an RSV promoter (SEQ ID NO: 3) produced the highest CSF progranulin levels. [0347] Delivery of the tested recombinant polynucleotides was evaluated by comparing copy numbers of viral genomes per diploid genome (vg/dg) in tissues extracted from mice. Viral genome copies were measured using primers and probes spanning GRN exon 7 and exon 8. Diploid genome copies were measured using mGAPDh primers and probes. In a first assay, vg/dg was quantified from DNA isolated using DNEAsy blood and tissue kit. FIG. 27A shows a plot comparing viral genomes per diploid genome (vg/dg) in brain tissue extracted from mice infected with scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6) (“scAAV9 RSV”), scAAV9 encapsidating a JeT promoter (SEQ ID NO: 4) operably linked to GRN coding sequence (SEQ ID NO: 6) (“scAAV9 JET”), scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6) and SV40 intron (SEQ ID NO: 13) (“scAAV9 RSV SV40 intron 99”), scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence containing GRN intron 9 (SEQ ID NO: 11) (“scAAV9 RSV intron 9”), ssAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6) (“ssAAV9 RSV”), ssAAV9 encapsidating a CAG promoter (SEQ ID NO: 1) operably linked to GRN coding sequence (SEQ ID NO: 6) (“ssAAV9 CAG”), ssAAV9 encapsidating a hSyn promoter (SEQ ID NO: 5) operably linked to GRN coding sequence (SEQ ID NO: 6) (“ssAAV9 hSyn”), or ssAAV9 encapsidating a CMV promoter (SEQ ID NO: 2) operably linked to GRN coding sequence (SEQ ID NO: 6) (“ssAAV9 CMV”). FIG. 27B shows a plot comparing viral genomes per diploid genome (vg/dg) in liver tissue extracted from mice infected with scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6) (“scAAV9 RSV”), scAAV9 encapsidating a JeT promoter (SEQ ID NO: 4) operably linked to GRN coding sequence (SEQ ID NO: 6) (“scAAV9 JET”), scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6) and SV40 intron (SEQ ID NO: 13) (“scAAV9 RSV SV40 intron 99”), scAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence containing GRN intron 9 (SEQ ID NO: 11) (“scAAV9 RSV intron 9”), ssAAV9 encapsidating an RSV promoter (SEQ ID NO: 3) operably linked to GRN coding sequence (SEQ ID NO: 6) (“ssAAV9 RSV”), ssAAV9 encapsidating a CAG promoter (SEQ ID NO: 1) operably linked to GRN coding sequence (SEQ ID NO: 6) (“ssAAV9 CAG”), ssAAV9 encapsidating a hSyn promoter (SEQ ID NO: 5) operably linked to GRN coding sequence (SEQ ID NO: 6) (“ssAAV9 hSyn”), or ssAAV9 encapsidating a CMV promoter (SEQ ID NO: 2) operably linked to GRN coding sequence (SEQ ID NO: 6) (“ssAAV9 CMV”). As shown in FIG. 27A, ssAAV9 RSV and scAAV9 RSV showed vg/dg levels in brain tissue that were higher than the controls.

[0348] In a second assay, vg/dg was quantified from DNA isolated using eluent from a MagMax Mirvana Total RNA kit without the Dnase treatment step. In this assay, AAV9 encapsidating a GRN coding sequence under transcriptional control of the RSV promoter (SEQ ID NO: 3) showed vg/dg levels in brain tissue that were higher than AAV9 encapsidating a GRN coding sequence under transcriptional control of a human synapsin promoter (SEQ ID NO: 5) as well as the controls.

[0349] Brain tissue is also assessed via histology. EXAMPLE 3

Effects of Progranulin Expression in Cultured Neurons

[0350] This example describes the effects of progranulin expression in cultured neuron models of frontotemporal dementia (FTD). GRN knockout neurons are grown in culture. Populations of neurons are infected with a virus containing a recombinant polynucleotide encoding progranulin under control of a promoter (any one of SEQ ID NO: 1 - SEQ ID NO: 5) or a control polynucleotide lacking a progranulin sequence. Neuron populations are evaluated for progranulin expression as well as downstream effects associated with progranulin levels, including caspase 3 activity, neurofilament formation, lysosome size, lipofuscin accumulation, microgliosis, and TDP43 accumulation.

[0351] An increase in progranulin expression is associated with a decrease in caspase 3 activity, indicative of improved neural cell health. An increase in progranulin expression is associated with a decrease in neuro filament light chain (NF-L) formation, indicative of a decrease in neuroinflammation, a FTD disease phenotype. An increase in progranulin expression is associated with a decrease in lysosome size, indicative of restored cellular activity in microglia and neurons. An increase in progranulin expression is associated with a decrease in lipofuscin accumulation, indicative of decreased lysosomal dysfunction. An increase in progranulin expression is associated with a decrease in TDP43 accumulation, an FTD disease marker.

EXAMPLE 4

Promoter Activity in Neuronal Cell Lines

[0352] This example describes activity of various promoters in two different neuronal cell lines. Promoter-driven expression was evaluated in both a human neuroblastoma cell line (SH-SY5Y cells) and a human neuroglioma cell line (H4 cells). A dual reporter plasmid, as illustrated in FIG. 12A, was generated for each of 11 promoters to be tested. Each dual reporter plasmid included a protein testing cassette containing a promoter (EFlα (SEQ ID NO: 22), a first synthetic promoter (Synth 1), a second synthetic promoter (Synth 2), a third synthetic promoter (Synth 3), a fourth synthetic promoter (Synth 4), JeT (SEQ ID NO: 4), MECP2 (SEQ ID NO: 23), RSV (SEQ ID NO: 3), YB-TATA (SEQ ID NO: 54), chicken B-actin (SEQ ID NO: 24), or MND (SEQ ID NO: 25)), a 5’ untranslated region, a sequence encoding mCherry, and a polyA signal, and a transfection control cassette containing a CMV promoter, a sequence encoding GFP, and a polyA signal. The JeT promoter had a length of 186 nucleotides, the RSV promoter had a length of 262 nucleotides, the chicken B-actin promoter had a length of 278 nucleotides, and the MND promoter had a length of 347 nucleotides. [0353] To evaluate promoter activity in neuroblastoma cells, each of the resulting 11 dual reporter plasmids and a pMax control plasmid were electroporated into approximately 40,000 SH-SY5Y cells. The cells were transfected with 200 ng of dual reporter plasmid DNA, and expression was measured via flow cytometry after 48 hours. Promoter activity was quantified as the ratio of mCherry fluorescence to GFP fluorescence, such that a higher mCherry/GFP ratio corresponded to higher promoter activity. As shown in FIG. 12B, the EF1α promoter, the JeT promoter, the RSV promoter, the chicken B-actin promoter, and the MND promoter exhibited robust expression in SH-SY5Y cells. The MECP2 also exhibited expression in SH-SY5Y cells. [0354] To evaluate promoter activity in neuroglioma cells, each of the dual reporter plasmids containing either the EFlα promoter, the JeT promoter, the MECP2 promoter, the RSV promoter, the chicken B-actin promoter, the MND promoter, or the pMax promoter were the electroporated into approximately 40,000 H4 cells. The cells were transfected with 200 ng of dual reporter plasmid DNA, and expression was measured via flow cytometry after 48 hours. Promoter activity was quantified as the ratio of mCherry fluorescence to GFP fluorescence, such that a higher mCherry/GFP ratio corresponded to higher promoter activity. As shown in FIG. 12C, the EFlα promoter, the JeT promoter, the RSV promoter, the chicken B-actin promoter, and the MND promoter exhibited robust expression in H4 cells. The MECP2 promoter also exhibited expression in H4 cells. The RSV promoter was selected for further development due to its compact size and high transcriptional activity in model cell lines, including CNS model cell lines.

EXAMPLE 5

Effect of Polyadenylation Signal and Untranslated Region on Payload Expression [0355] This example describes the effect of various polyadenylation (poly A) signals and untranslated regions on payload expression. A reporter plasmid containing a luciferase expression cassette, as shown in FIG. 13A, was generated for each of nine polyA signal sequences (SEQ ID NO: 40 - SEQ ID NO: 48). Each expression cassette contained a CMV promoter, a sequence encoding Sec. luciferase, and a polyA signal sequence (one of SEQ ID NO: 40 - SEQ ID NO: 48). Rabbit beta globin polyA (SEQ ID NO: 42) had a length of 56 nucleotides.

[0356] Each of the resulting 9 plasmids and a pMax control plasmid were transfected into approximately 40,000 HEK293 cells using lipid nanoparticle transfection. The cells were transfected with 500 ng of plasmid DNA, and expression was measured via luminescence after 48 hours. Water (“H2O”) was used as a negative control. Higher luminescence corresponded to higher expression. As shown in FIG. 13B, the rabbit beta globin polyA (SEQ ID NO: 42) exhibited the highest expression. The paired bars represent biological replicates.

EXAMPLE 6

Progranulin Expression with Inclusion of a GRN Intron

[0357] This example describes the effect of various GRN introns on progranulin expression and the increase in progranulin expression with the inclusion of GRN intron 9. An expression plasmid, as illustrated in FIG. 14A, was generated for each of the three GRN introns to be tested paired with each of two promoters to be tested. Each expression plasmid included a GRN expression cassette containing either a JeT a promoter (SEQ ID NO: 4) or an RSV promoter (SEQ ID NO: 3), a 5’ untranslated region, a progranulin coding sequence containing one of three endogenous GRN introns (intron 5, intron 9, intron 11, or intron 12) or no intron, and a polyA signal, and a normalization expression cassette containing a CMV promoter, a sequence encoding a secreted luciferase (“Sec. luciferase”), and a polyA signal. The nine resulting expression plasmids contained GRN expression cassettes with either a JeT promoter (SEQ ID NO: 4) and a human progranulin coding sequence (SEQ ID NO: 6) with no introns (“Jet”), an RSV promoter (SEQ ID NO: 3) and a human progranulin coding sequence (SEQ ID NO: 6) with no introns (“RSV”), a JeT promoter (SEQ ID NO: 4) and a human progranulin coding sequence with GRN intron 5 (SEQ ID NO: 27; “Jet intron 5”), a JeT promoter (SEQ ID NO: 4) and a human progranulin coding sequence with GRN intron 9 (SEQ ID NO: 12; “Jet intron 9”), a JeT promoter (SEQ ID NO: 4) and a human progranulin coding sequence with GRN intron 12 (SEQ ID NO: 29; “Jet intron 12”), an RSV promoter (SEQ ID NO: 3) and a human progranulin coding sequence with GRN intron 5 (SEQ ID NO: 27; “RSV intron 5”), an RSV promoter (SEQ ID NO: 3) and a human progranulin coding sequence with GRN intron 9 (SEQ ID NO: 12; “RSV intron 9”), an RSV promoter (SEQ ID NO: 3) and a human progranulin coding sequence with GRN intron 11 (SEQ ID NO: 29; “RSV intron 11”), or an RSV promoter (SEQ ID NO: 3) or a human progranulin coding sequence with GRN intron 12 (SEQ ID NO: 29; “RSV intron 12”). [0358] To evaluate the effect of introns on progranulin expression levels, each of the resulting nine expression plasmids were electroporated into approximately 40,000 cells. The cells were transfected with 500 ng of plasmid DNA and assayed after 48 hours. Progranulin expression was measured via ELISA and was normalized to luciferase luminescence to control for transfection efficiency. As shown in FIG. 14B, the progranulin expression cassette containing an RSV promoter (SEQ ID NO: 3) and a progranulin coding sequence with intron 9 (SEQ ID NO: 11) exhibited the highest expression. EXAMPLE 7 Improved Progranulin Expression with Inclusion of a Post-Transcriptional Regulatory

Element

[0359] This example describes the effect of a post-transcriptional regulatory element on progranulin expression. A full length WPRE and a truncated WPRE (“WPRE3”) were tested, and an increase in progranulin expression was observed with the inclusion of either the full length WPRE or the WPRE3. An expression plasmid, as illustrated in FIG. 17A, was generated for inclusion of a full length WPRE (“WPRE”) or a truncated WPRE (“WPRE3”) to be tested, or no WPRE or WPRE3. Each expression plasmid included a GRN expression cassette containing an RSV promoter, a progranulin coding sequence (“progranulin CDS”), optionally, WPRE (SEQ ID NO: 7) or WPRE3 (SEQ ID NO: 8), and a rabbit beta globin polyadenylation signal (“RBG Poly A”; SEQ ID NO: 42) and a normalization expression cassette containing a CMV promoter (SEQ ID NO: 2), a luciferase coding sequence (“Sec. Luciferase”), and a polyadenylation signal (“polyA sig”). More specifically, a first plasmid tested had a GRN expression cassette, wherein the sequence of the progranulin CDS was SEQ ID NO: 10 and there was no WPRE or WPRE3 sequence (“-”); a second plasmid tested had a GRN expression cassette, wherein the sequence of progranulin CDS was SEQ ID NO: 10 and the sequence of WPRE was SEQ ID NO: 7 (“WPRE”); a third plasmid tested had a GRN expression cassette, wherein the sequence of progranulin CDS was SEQ ID NO: 10 and the sequence of WPRE3 was SEQ ID NO: 8 (“WPRE3”); a fourth plasmid tested had a GRN expression cassette, wherein the sequence of progranulin CDS was SEQ ID NO: 14 and no WPRE or WPRE3 sequence (“SV40in99-”); a fifth plasmid tested had a GRN expression cassette, wherein the sequence of progranulin CDS was SEQ ID NO: 14 and the sequence of WPRE was SEQ ID NO: 7 (“SV40int99 WPRE”); a sixth plasmid tested had a GRN expression cassette, wherein the sequence of progranulin CDS was SEQ ID NO: 14 and the sequence of WPRE was SEQ ID NO: 8 (“SV40int99WPRE3”); and a seventh plasmid tested had a GRN expression cassette, wherein the sequence of progranulin CDS was SEQ ID NO: 11 and the sequence of WPRE was SEQ ID NO: 8 (“endo GRNint9 WPRE”).

[0360] FIG. 17B shows ELISA data of progranulin expression fold change normalized to luciferase activity from H4 GRN KO cells transfected with the expression plasmid of [0361] To evaluate the effect of WPRE or WPRE3 on progranulin expression levels, each of the resulting seven expression plasmids were electroporated into approximately H4 GRN KO cells. The cells were transfected with 600 ng of plasmid DNA and then assayed for progranulin expression. Progranulin expression was measured via ELISA and was normalized to luciferase luminescence to control for transfection efficiency. As shown in FIG. 17B, the progranulin expression cassette containing a WPRE3 (SEQ ID NO: 8) exhibited the highest expression. [0362] The effect of WPRE or WPRE3 on progranulin expression levels was further evaluated in in human neuronal induced pluripotent stem cells (iPSCs, FIG. 18A) and mouse primary neurons (FIG. 18B). Expression constructs were delivered to the iPSCs or mouse primary neurons using AAV vectors. Each AAV included a GRN expression cassette containing an RSV promoter (SEQ ID NO: 3), a progranulin coding sequence, either a WPRE (SEQ ID NO: 7) or WPRE3 (SEQ ID NO: 8), and a rabbit beta globin polyadenylation signal (SEQ ID NO: 42) and a normalization expression cassette containing a CMV promoter (SEQ ID NO: 2), a luciferase coding sequence, and a polyadenylation signal. More specifically, a first AAV tested had a GRN expression cassette with a WPRE of SEQ ID NO: 7 (“RSV + WPRE”) and a second AAV tested had a GRN expression cassette with a WPRE3 of SEQ ID NO: 8 (“RSV + WPRE3”). Progranulin expression levels were measured on days 3 and 7 following AAV delivery. As shown in FIG. 18A and FIG. 18B, progranulin was successfully expressed for both WPRE and WPRE3 constructs in both iPSCs and mouse primary neurons. The full length WPRE (SEQ ID NO: 7) and truncated WPRE (WPRE3, SEQ ID NO: 8) produced similar levels of progranulin. The truncated element (WPRE3) was selected for further construct development for its smaller size compared to the full length WPRE.

EXAMPLE 8

Progranulin Expression with Inclusion of an SV40 Intron in the 5’ Untranslated Region [0363] This example describes the effect of various introns in the 5 ’ untranslated region (5 ’ UTR) on progranulin expression and the increase in progranulin expression with the inclusion of an SV40 intron. An expression plasmid, as illustrated in FIG. 15A, was generated for each of the six introns to be tested or no intron. Each expression plasmid included a GRN expression cassette containing an RSV promoter (SEQ ID NO: 3), a 5’ untranslated region containing one of six introns (GRN11 (SEQ ID NO: 28), GRN12 (SEQ ID NO: 29), GRN9 (SEQ ID NO: 12), SV40-99 (SEQ ID NO: 13), SVsmT (SEQ ID NO: 30), or TP13 (SEQ ID NO: 31)) or no intron, a progranulin coding sequence, and a polyA signal, and a normalization expression cassette containing a CMV promoter, a sequence encoding Sec. luciferase, and a polyA signal. [0364] To evaluate the effect of introns within the 5’ untranslated region on progranulin expression levels, each of the resulting seven expression plasmids were electroporated into approximately 40,000 HEK cells. The cells were transfected with 500 ng of plasmid DNA and assayed after 48 hours. Progranulin expression was measured via ELISA and was normalized to luciferase luminescence to control for transfection efficiency. As shown in FIG. 15B, the progranulin expression cassette containing an SV40-99 intron (SEQ ID NO: 13) in the 5’ untranslated region exhibited the highest expression in HEK cells. Circles and triangles in FIG. 15B show a first replicate and a second replicate, respectively, of each assay.

[0365] The effects of including an SV40 intron in the 5’UTR or a intron 9 in the progranulin coding sequence (GRN intron9) were further tested in a human neuroglioma (H4) cells with a progranulin knockout. As shown in FIG. 25A, expression cassettes including a progranulin coding sequence under control of an RSV promoter (SEQ ID NO: 3), RSV and an SV40 intron of SEQ ID NO: 13 in the 5’UTR (“RSV_SV40intron”), or RSV and a progranulin coding sequence comprising a progranulin intron 9 of SEQ ID NO: 12 (“RSV_endo_intm9”) were delivered to H4 progranulin knockout cells using scAAV vectors, and secreted progranulin levels were measured. An expression cassette including a progranulin coding sequence under control of a JeT promoter (SEQ ID NO: 4) (“JeT”) was also tested. As shown in FIG. 25B, AAV plasmids comprising expression cassettes including a progranulin coding sequence under control of an RSV promoter (SEQ ID NO: 3) (“scAAV RSV”) or RSV and an SV40 intron of SEQ ID NO: 13 in the 5’UTR (“scAAV_RSV_SV40intron99”) were transfected into H4 progranulin knockout cells, and secreted progranulin levels were measured. Expression cassettes delivered including progranulin coding sequences under control of either an RSV promoter of SEQ ID NO: 3 (“ssAAV_RSV”), a CAG promoter of SEQ ID NO: 1 (“ssAAV_CAG”), a synapsin promoter of SEQ ID NO: 5 (“ssAAV hSyn”), or a CMV promoter of SEQ ID NO: 2 (“ssAAV_CMV”) were also tested. As seen in FIG. 25A and FIG. 25B, inclusion of an SV40 intron or a progranulin intron9 did not enhance progranulin production in H4 progranulin knockout cells compared to an RSV promoter and a progranulin coding sequence without introns.

EXAMPLE 9 Manufacturing rAAVs Encapsidating a Sequence Coding for Progranulin from a Stable Cell Line

[0366] This example describes manufacturing recombinant AAVs (rAAVs) encapsidating a sequence coding for progranulin from a stable cell line. A stable mammalian cell line capable of inducible expression of rAAVs encapsidating a sequence encoding progranulin was constructed. Briefly, a suspension culture of HEK 293 cells was transfected with a plasmid for inducible expression of adenoviral helper genes with a selectable marker for puromycin resistance. Following puromycin selection, a Pl cell line was established. The Pl cell line was transfected with two plasmids: a plasmid encoding inducible AAV Rep and Cap proteins and a first portion of a selectable marker for blasticidin resistance; and a plasmid encoding a progranulin (PGRN) payload (SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, or SEQ ID NO: 74) and a second portion of the selectable marker for blasticidin resistance. Blasticidin selection was then introduced on day 3 post transfection and cells were passaged in selective media until they recovered. All pools were banked and analyzed for production of rAAV5 after induction with Dox and Tamoxifen.

[0367] The following four PGRN payload constructs were tested for viral titer and ecapsidation: GRN under transcriptional control of a Jet promoter (plasmid sequence SEQ ID NO: 72, ITR to ITR sequence SEQ ID NO: 68, stable cell pool T204); GRN under transcriptional control of a RSV promoter (plasmid sequence SEQ ID NO: 71, ITR to ITR sequence SEQ ID NO: 67, stable cell pool T205); GRN with intron9 under transcriptional control of a RSV Promoter (plasmid sequence SEQ ID NO: 73, ITR to ITR sequence SEQ ID NO: 69, stable cell pool T206); and GRN under transcriptional control of a RSV Promoter with an SV40 intron (plasmid sequence SEQ ID NO: 74, ITR to ITR sequence SEQ ID NO: 70, stable cell pool T207). A control cell line with a mock transfection was also generated (stable cell pool T209).

[0368] Stable cell pools (T204, T205, T206, and T207) harboring one of the four different payload constructs were analyzed for viable cell density and percent viability (FIG. 19) and for rAAV encapsidating a nucleic acid sequence encoding PGRN (vg/mL) and rAAV capsid (vp/mL) titers (FIG. 20).

[0369] The stable cell pool T205, containing a PGRN payload construct with an RSV promoter, was selected for single cell cloning. T205 cells were seeded at single cell density and expanded. Seventy expanded single cell clones were analyzed for viral genome (vg), Rep, and Cap titers.

[0370] The data shown in FIG. 21 is provided in TABLE 9.

TABLE 9. Packaging Data

[0371] Furthermore, as shown in FIG. 22, the titer produced by the top single cell clone with a progranulin payload under control of an RSV promoter (“Clone D (PGRN)”) showed a greater than 10-fold improvement over transient transfection titer. Furthermore, cell clones from the stable cell line having a progranulin payload under control of an RSV promoter (“T205 Pool (PGRN)”, “Clone A (PGRN)”, “Clone B (PGRN)”, “Clone C (PGRN)”, and “Clone D (PGRN)”) showed comparable capsid titers to stable cell lines having an eGFP payload (“T42 Pool (eGFP)”, “Clone A (eGFP)”, “Clone B (eGFP)”, “Clone C (eGFP)”, and “Clone D (eGFP)”), demonstrating that AAVs with a progranulin payload can be successfully manufactured from a stable cell line with good viral titers and payload encapsidation. Titer was measured from AAV5-sc-payload lysate pre-purification by capsid ELISA for viral particle. [0372] FIG. 23 shows progranulin production (PGRN pg/mL) by cells infected at the indicated multiplicity of infection of rAAV virion titers (MOI vg/cell) that were produced after induction of a single cell clone cell line (CL23) selected from the stable cell line pool T205. This data demonstrates that the rAAV virions produced by a stable cell single cell clone line were infectious and produced a progranulin (PGRN) payload. Together, these data demonstrate the successful manufacturing of rAAV virions with a progranulin payload from a stable cell line. [0373] An additional stable cell line is generated with a plasmid encoding AAV Rep and Cap proteins and a plasmid of SEQ ID NO: 120 encoding a progranulin (PGRN) payload comprising an ITR to ITR sequence of SEQ ID NO: 65. The stable cell line is generated and is evaluated using the methods described above, showing this stable cell line is capable of generating good viral titer, high payload encapsidation (encapsidation of the progranulin payload of SEQ ID NO: 65), infectious virion, and secreted progranulin after infection with the generated virion.

EXAMPLE 10

In Vivo Expression of Progranulin in Mice

[0374] This example describes in vivo expression of progranulin in mice. Mice were injected via intracerebro ventricular (ICV) injection with AAV9 virions containing expression cassettes encoding human progranulin under transcriptional control of an RSV promoter or a human synapsin promoter. RNA transcription levels of human progranulin (GRN/GAPDH) were compared between the RSV promoter (“RSV”) and the human synapsin promoter (“hSyn”) in brain tissue (FIG. 24A). As shown in FIG. 24A, the expression cassette with the RSV promoter (SEQ ID NO: 65) produced higher human progranulin RNA expression in the brain than the expression cassette with the human synapsin promoter.

[0375] Human progranulin protein levels (“hPGRN protein level” on y-axis; ng/ml) were also compared between expression cassettes encoding human progranulin under transcriptional control of either the RSV promoter (“RSV”) or the human synapsin promoter (“hSyn”) in cerebrospinal fluid (CSF) (FIG. 24B). As shown in FIG. 24B, the expression cassette with the RSV promoter (SEQ ID NO: 65) produced higher human progranulin protein levels in cerebrospinal fluid (CSF) (“hPGRN protein level” on y-axis; ng/ml) than the expression cassette with the human synapsin promoter.

EXAMPLE 11

Improved Progranulin Expression with Inclusion of a Transcriptional Pause Site [0376] This example describes the effect of a transcriptional pause site on progranulin expression. Expression plasmids including a normalization expression cassette encoding luciferase and a progranulin expression cassette (e.g., having SEQ ID NO: 65) encoding progranulin under control of an RSV promoter, with or without a transcriptional pause site of SEQ ID NO: 64, and expression plasmids (e.g., having SEQ ID NO: 65) encoding a progranulin expression cassette encoding progranulin under control of an RSV promoter, with or without a transcriptional pause site of SEQ ID NO: 64 are tested. These plasmids are transfected into H4 progranulin knockout cells, and secreted progranulin protein levels are measured in the cell supernatant. Progranulin expression is higher for expression plasmids having the transcriptional pause site than for expression plasmids without the transcriptional pause site. It is expected that the transcriptional pause site increases progranulin expression by reducing transcriptional silencing due to transcriptional readthrough from the upstream promoter.

[0377] This example describes the effect of a transcriptional pause site on progranulin expression. AAV containing an expression cassette including a normalization expression cassette encoding luciferase and a progranulin expression cassette (e.g., having SEQ ID NO: 65) encoding progranulin under control of an RSV promoter, with or without a transcriptional pause site of SEQ ID NO: 64, and expression plasmids encoding a progranulin expression cassette (e.g., having SEQ ID NO: 65) encoding progranulin under control of an RSV promoter, with or without a transcriptional pause site of SEQ ID NO: 64 are tested. These AAV are injected intravenously into mice and secreted progranulin protein levels are measured in the serum and CSF. Progranulin expression is higher for expression cassettes having the transcriptional pause site than for expression plasmids without the transcriptional pause site. It is expected that the transcriptional pause site increases progranulin expression by reducing transcriptional silencing in vivo.

EXAMPLE 12

In vitro and Ex Vivo Expression of Progranulin in Cultured Cells

[0378] This example describes in vitro and ex vivo expression of progranulin in various cultured cell lines. Cultured cells are transfected with progranulin expression cassette encoding human progranulin under transcriptional control of an RSV promoter (SEQ ID NO: 65). The cultured cells are H4 progranulin knockout cells, induced pluripotent stem cells (iPSCs) derived neurons of human patients having a condition associated with progranulin or a progranulin knockout line, or mouse primary neurons from wild type and progranulin knockout mice. Secreted progranulin protein levels, measured in the supernatant of the cultured cells, are evaluated. Progranulin concentrations in the cell supernatant are comparable to or greater than those produced by wild type cells expressing endogenous progranulin are targeted.

[0379] Additional in vitro and ex vivo expression of progranulin in various cultured cell lines are tested. Ex vivo cultures are of primary mouse neurons (wild-type background). The in vitro cultures are of progranulin knockout human iPSC-derived neurons (wild-type GRN background engineered to knock-out both GRN copies) and a HEPG2 cell line. Secreted progranulin protein levels, measured in the supernatant of the cultured cells, are evaluated. Progranulin concentrations in the cell supernatant comparable to or greater than those produced by wild type cells expressing endogenous progranulin are targeted.

EXAMPLE 13

In Vivo Expression of Progranulin in Progranulin Knockout Mice

[0380] This example describes in vivo expression of progranulin that secretes into the cerebrospinal fluid of progranulin knockout mice. Experiments are performed in wild type, 4- month-old progranulin knockout mice, and 10-month-old progranulin knockout mice. PhP.eB AAVs containing an expression cassette encoding human progranulin under transcriptional control of an RSV promoter (SEQ ID NO: 65) are generated. The AAVs are administered to progranulin knockout mice via tail vein injection, and progranulin protein levels are measured in cerebrospinal fluid (CSF) and serum. For short term studies, progranulin protein levels are measured in cerebrospinal fluid (CSF) extracted from the mice at 4 weeks and serum extracted from the mice at 2 and 4 weeks following AAV administration. For long term studies, progranulin protein levels are measured in cerebrospinal fluid (CSF) extracted from the mice at 2, 3, or 4 months weeks and serum extracted from the mice at 2 weeks and 2, 3, or 4 months following AAV administration. Progranulin levels in CSF of 10 ng/mL to 50 ng/mL are targeted. Progranulin levels in serum of less than 300 ng/mL are targeted. AAV dosing is adjusted to achieve target progranulin expression levels in CSF and serum.

EXAMPLE 14

Expression of Progranulin in Cerebrospinal Fluid of Non-Human Primates [0381] This example describes expression of progranulin in cerebrospinal fluid of non-human primates. Engineered AAV5 virions assembled from VP capsid polypeptides comprising a 581- 589 region sequence of SEQ ID NO: 83 and encapsidating an expression cassette encoding human progranulin under transcriptional control of an RSV promoter (SEQ ID NO: 65) are generated, optionally using a stable cell line as described in EXAMPLE 9. The expression cassette encodes human progranulin under transcriptional control of an RSV promoter (SEQ ID NO: 3). The engineered AAV5 virions are intravenously administered to a non-human primate, and progranulin protein levels are measured in cerebrospinal fluid (CSF) and serum extracted from the non-human primate weekly for eight weeks following AAV administration. Human progranulin is detected in CSF and serum. AAV dosing is adjusted to achieve a target progranulin expression level in CSF and a target progranulin expression level in serum. Tissue distribution is also assessed.

EXAMPLE 15

Expression of Progranulin in Cerebrospinal Fluid of Non-Human Primates [0382] This example describes expression of progranulin in cerebrospinal fluid of non-human primates. Engineered AAV5 virions assembled from VP capsid polypeptides comprising a 581- 589 region sequence of SEQ ID NO: 84 and encapsidating an expression cassette encoding human progranulin under transcriptional control of an RSV promoter (SEQ ID NO: 65) are generated, optionally using a stable cell line as described in EXAMPLE 9. The expression cassette encodes human progranulin under transcriptional control of an RSV promoter (SEQ ID NO: 3). The engineered AAV5 virions are intravenously administered to a non-human primate, and progranulin protein levels are measured in cerebrospinal fluid (CSF) and serum extracted from the non-human primate weekly for eight weeks following AAV administration. Human progranulin is detected in CSF and serum. AAV dosing is adjusted to achieve a target progranulin expression level in CSF and a target progranulin expression level in serum. Tissue distribution is also assessed. EXAMPLE 16

Expression of Progranulin in Cerebrospinal Fluid of Non-Human Primates

[0383] This example describes expression of progranulin in cerebrospinal fluid of non-human primates. Engineered AAV5 virions assembled from VP capsid polypeptides comprising a 581- 589 region sequence of any one of SEQ ID NO: 82 or SEQ ID NO: 85 - SEQ ID NO: 111 and encapsidating an expression cassette encoding human progranulin under transcriptional control of an RSV promoter (SEQ ID NO: 65) are generated, optionally using a stable cell line as described in EXAMPLE 9. The expression cassette encodes human progranulin under transcriptional control of an RSV promoter (SEQ ID NO: 3). The engineered AAV5 virions are intravenously administered to a non-human primate, and progranulin protein levels are measured in cerebrospinal fluid (CSF) and serum extracted from the non-human primate weekly for eight weeks following AAV administration. Human progranulin is detected in CSF and serum. AAV dosing is adjusted to achieve a target progranulin expression level in CSF and a target progranulin expression level in serum. Tissue distribution is also assessed.

EXAMPLE 17

Treatments of Frontotemporal Dementia (FTD)

[0384] This example describes various treatments for a subject having frontotemporal dementia (FTD) by delivering a recombinant polynucleotide of the present disclosure. Five different recombinant polynucleotides that can each individually be used for treatment is described. [0385] An AAV containing a recombinant polynucleotide having a CAG promoter operably linked to a sequence encoding progranulin is delivered to a subject. The subject is exhibiting symptoms of Frontotemporal Dementia and/or has been diagnosed with frontotemporal dementia. After administration of the AAV containing the recombinant polynucleotide, progranulin is expressed from the recombinant polynucleotide. The subject suffering symptoms of frontotemporal dementia thus experiences a reduction in symptoms following treatment. [0386] An AAV containing a recombinant polynucleotide having a CMV promoter operably linked to a sequence encoding progranulin is delivered to a subject. The subject is exhibiting symptoms of Frontotemporal Dementia and/or has been diagnosed with frontotemporal dementia. After administration of the AAV containing the recombinant polynucleotide, progranulin is expressed from the recombinant polynucleotide. The subject suffering symptoms of frontotemporal dementia thus experiences a reduction in symptoms following treatment.

[0387] Optionally, the recombinant polynucleotide is SEQ ID NO: 65.

[0388] An AAV containing a recombinant polynucleotide having an RSV promoter operably linked to a sequence encoding progranulin is delivered to a subject. The subject is exhibiting symptoms of Frontotemporal Dementia and/or has been diagnosed with frontotemporal dementia. After administration of the AAV containing the recombinant polynucleotide, progranulin is expressed from the recombinant polynucleotide. The subject suffering symptoms of frontotemporal dementia thus experiences a reduction in symptoms following treatment. [0389] An AAV containing a recombinant polynucleotide having a hSynapsin promoter operably linked to a sequence encoding progranulin is delivered to a subject. The subject is exhibiting symptoms of Frontotemporal Dementia and/or has been diagnosed with frontotemporal dementia. After administration of the AAV containing the recombinant polynucleotide, progranulin is expressed from the recombinant polynucleotide. The subject suffering symptoms of frontotemporal dementia thus experiences a reduction in symptoms following treatment.

EXAMPLE 18

Treatments of Frontotemporal Dementia (FTD) with a Recombinant Polynucleotide Encoding Progranulin Under Control of an RSV Promoter

[0390] This example describes various treatments for a subject having frontotemporal dementia (FTD) by delivering a recombinant polynucleotide of the present disclosure that encodes progranulin under control of an RSV promoter.

[0391] An AAV containing a recombinant polynucleotide having an RSV promoter operably linked to a sequence encoding progranulin is delivered to a subject. Optionally, the RSV promoter has a sequence of SEQ ID NO: 3. The recombinant polynucleotide further includes a post-transcriptional regulatory element; optionally the post-transcriptional regulatory element is a WPRE3 having a sequence of SEQ ID NO: 8. The recombinant polynucleotide further includes a polyadenylation signal; optionally the polyadenylation signal is a rabbit beta globin polyA signal having a sequence of SEQ ID NO: 42. The recombinant polynucleotide further includes a transcriptional pause site; optionally the transcriptional pause site is a synthetic transcriptional pause site with an additional polyA signal having a sequence of SEQ ID NO: 64. Optionally, the recombinant polynucleotide comprises a sequence of SEQ ID NO: 65. The subject is exhibiting symptoms of Frontotemporal Dementia and/or has been diagnosed with frontotemporal dementia. After administration of the AAV containing the recombinant polynucleotide, progranulin is expressed from the recombinant polynucleotide. The progranulin is expressed in a cerebrospinal fluid of the subject at a therapeutic level comparable to that of a healthy patient. Optionally, the progranulin is expressed in the cerebrospinal fluid of the subject at 70 ng/mL to 180 ng/mL or 70 ng/mL to 300 ng/mL. The subject suffering symptoms of frontotemporal dementia thus experiences a reduction in symptoms following treatment. [0392] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A recombinant polynucleotide comprising: an RSV promoter; a coding sequence operably linked to the RSV promoter, wherein the coding sequence encodes a progranulin; a post-transcriptional regulatory element; a polyadenylation signal; and a transcriptional pause site.

2. The recombinant polynucleotide of claim 1, wherein the post-transcriptional regulatory element comprises a WPRE.

3. The recombinant polynucleotide of claim 2, wherein the WPRE is a shortened WPRE.

4. The recombinant polynucleotide of claim 3, wherein the shortened WPRE is a WPRE3 comprising at least at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 8.

5. The recombinant polynucleotide of claim 3 or claim 4, wherein the WPRE3 comprises at least 90% sequence identity to SEQ ID NO: 8.

6. The recombinant polynucleotide of any one of claims 3-5, wherein the WPRE3 comprises a sequence of SEQ ID NO: 8.

7. The recombinant polynucleotide of claim 2, wherein the WPRE comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 7.

8. The recombinant polynucleotide of claim 7, wherein the WPRE comprises at least 90% sequence identity to SEQ ID NO: 7.

9. The recombinant polynucleotide of claim 7 or claim 8, wherein the WPRE comprises a sequence of SEQ ID NO: 7.

10. The recombinant polynucleotide of any one of claims 1-9, wherein the post- transcriptional regulatory element is downstream of the coding sequence.

11. The recombinant polynucleotide of any one of claims 1-10, wherein the polyadenylation signal comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 42, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 43 - SEQ ID NO: 48, or SEQ ID NO: 55.

12. The recombinant polynucleotide of any one of claims 1-11, wherein the polyadenylation signal comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 42.

13. The recombinant polynucleotide of any one of claims 1-12, wherein the polyadenylation signal comprises at least 90% sequence identity to SEQ ID NO: 42.

14. The recombinant polynucleotide of any one of claims 1-12, wherein the polyadenylation signal comprises a sequence of SEQ ID NO: 42.

15. The recombinant polynucleotide of any one of claims 1-14, wherein the polyadenylation signal is downstream of the coding sequence.

16. The recombinant polynucleotide of any one of claims 1-15, wherein the transcriptional pause site comprises a second polyadenylation signal.

17. The recombinant polynucleotide of any one of claims 1-16, wherein the transcriptional pause site comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 64.

18. The recombinant polynucleotide of any one of claims 1-17, wherein the transcriptional pause site comprises at least 90% sequence identity to SEQ ID NO: 64.

19. The recombinant polynucleotide of any one of claims 1-18, wherein the transcriptional pause site comprises a sequence of SEQ ID NO: 64.

20. The recombinant polynucleotide of any one of claims 1-19, wherein the transcriptional pause site is downstream of the coding sequence.

21. The recombinant polynucleotide of any one of claims 1-20, wherein the transcriptional pause site is downstream of the polyadenylation signal.

22. The recombinant polynucleotide of any one of claims 1-21, wherein the coding sequence is downstream of the RSV promoter, the post-transcriptional regulatory element is downstream of the coding sequence, the polyadenylation signal is downstream of the post-transcriptional regulatory element, and the transcriptional pause site is downstream of the post-transcriptional regulatory element.

23. The recombinant polynucleotide of any one of claims 1-22, wherein the coding sequence is downstream of the RSV promoter, the post-transcriptional regulatory element is downstream of the coding sequence, the polyadenylation signal is downstream of the post-transcriptional regulatory element, and the transcriptional pause site is downstream of the polyadenylation signal.

24. The recombinant polynucleotide of any one of claims 1-23, wherein the RSV promoter comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 3.

25. The recombinant polynucleotide of any one of claims 1-24, wherein the RSV promoter comprises a sequence of SEQ ID NO: 3.

26. The recombinant polynucleotide of any one of claims 1-25, wherein the progranulin is a human progranulin.

27. The recombinant polynucleotide of any one of claims 1-26, wherein the progranulin coding sequence codes for an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 63.

28. The recombinant polynucleotide of any one of claims 1-27, wherein the progranulin coding sequence codes for an amino acid sequence comprising the sequence of SEQ ID NO: 63.

29. The recombinant polynucleotide of any one of claims 1-28, wherein the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 10.

30. The recombinant polynucleotide of any one of claims 1-29, wherein the coding sequence comprises a sequence of SEQ ID NO: 10.

31. The recombinant polynucleotide of any one of claims 1-29, wherein the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 6.

32. The recombinant polynucleotide of any one of claims 1-29, wherein the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 11.

33. The recombinant polynucleotide of any one of claims 1-, wherein the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 26.

34. The recombinant polynucleotide of any one of claims 1-, wherein the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 57 and at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 58.

35. The recombinant polynucleotide of any one of claims 1-34, further comprising a stuffer sequence.

36. The recombinant polynucleotide of claim 35, wherein the stuffer sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 61.

37. The recombinant polynucleotide of claim 35 or 36, wherein the stuffer sequence comprises a sequence of SEQ ID NO: 61.

38. The recombinant polynucleotide of any one of claims 35-37, wherein the stuffer sequence is upstream of the coding sequence.

39. The recombinant polynucleotide of any one of claims 35-38, wherein the stuffer sequence is upstream of the RSV promoter.

40. The recombinant polynucleotide of any one of claims 35-39, wherein the stuffer sequence is upstream of the RSV promoter, the coding sequence is downstream of the RSV promoter, the post-transcriptional regulatory element is downstream of the coding sequence, the polyadenylation signal is downstream of the post-transcriptional regulatory element, and the transcriptional pause site is downstream of the polyadenylation signal.

41. The recombinant polynucleotide of any one of claims 1-40, comprising a 5’ untranslated region.

42. The recombinant polynucleotide of claim 41, wherein the 5’ untranslated region comprises a Kozak sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 66.

43. The recombinant polynucleotide of claim 41 or claim 42, wherein the 5 ’ untranslated region comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 56.

44. The recombinant polynucleotide of any one of claims 41-43, wherein the 5 ’ untranslated region comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 62.

45. The recombinant polynucleotide of any one of claims 41-44, wherein the 5 ’ untranslated region comprises a sequence of SEQ ID NO: 62.

46. The recombinant polynucleotide of any one of claims 41-45, wherein the 5 ’ untranslated region is upstream of the coding sequence.

47. The recombinant polynucleotide of any one of claims 41-46, wherein the 5 ’ untranslated region is downstream of the RSV promoter.

48. The recombinant polynucleotide of any one of claims 41-47, wherein the stuffer sequence is upstream of the RSV promoter, the 5 ’ untranslated region is downstream of the RSV promoter, the coding sequence is downstream of the 5 ’ untranslated region, the post- transcriptional regulatory element is downstream of the coding sequence, the polyadenylation signal is downstream of the post-transcriptional regulatory element, and the transcriptional pause site is downstream of the polyadenylation signal.

49. The recombinant polynucleotide of any one of claims 1-48, further comprising a 5’ inverted terminal repeat upstream of the RSV promoter and a 3 ’ inverted terminal repeat downstream of the transcriptional pause site.

50. The recombinant polynucleotide of any one of claims 1-48, further comprising a 5’ inverted terminal repeat upstream of the stuffer sequence and a 3 ’ inverted terminal repeat downstream of the transcriptional pause site.

51. The recombinant polynucleotide of any one of claims 1-48, further comprising a 5’ inverted terminal repeat and a 3 ’ inverted terminal repeat, wherein the 5 ’ inverted terminal repeat is upstream of the stuffer sequence, the stuffer sequence is upstream of the RSV promoter, the 5 ’ untranslated region is downstream of the RSV promoter, the coding sequence is downstream of the 5 ’ untranslated region, the post-transcriptional regulatory element is downstream of the coding sequence, the polyadenylation signal is downstream of the post- transcriptional regulatory element, the transcriptional pause site is downstream of the polyadenylation signal, and the 3 ’ inverted terminal repeat downstream of the transcriptional pause site.

52. The recombinant polynucleotide of any one of claims 49-51 , wherein the 5 ’ inverted terminal repeat is an AAV 5 ’ inverted terminal repeat.

53. The recombinant polynucleotide of claim 52, wherein the AAV 5 ’ inverted terminal repeat is a single stranded AAV 5 ’ inverted terminal repeat.

54. The recombinant polynucleotide of any one of claims 49-53, wherein the 5 ’ inverted terminal repeat is an AAV5 5’ inverted terminal repeat, an AAV1 5’ inverted terminal repeat, an AAV2 5’ inverted terminal repeat, an AAV9 5’ inverted terminal repeat, or a PhP.eB 5’ inverted terminal repeat.

55. The recombinant polynucleotide of claim 49-54, wherein the 5’ inverted terminal repeat is an AAV2 5’ inverted terminal repeat.

56. The recombinant polynucleotide of claim 49-55, wherein the 5’ inverted terminal repeat is a single stranded AAV2 5’ inverted terminal repeat.

57. The recombinant polynucleotide of any one of claims 49-56, wherein the 5 ’ inverted terminal repeat comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 59.

58. The recombinant polynucleotide of any one of claims 49-57, wherein the 5 ’ inverted terminal repeat comprises a sequence of SEQ ID NO: 59.

59. The recombinant polynucleotide of any one of claims 49-58, wherein the 3 ’ inverted terminal repeat is an AAV 3 ’ inverted terminal repeat.

60. The recombinant polynucleotide of claim 59, wherein the AAV 3 ’ inverted terminal repeat is a single stranded AAV 3 ’ inverted terminal repeat.

61. The recombinant polynucleotide of any one of claims 49-60, wherein the 3 ’ inverted terminal repeat is an AAV5 3’ inverted terminal repeat, an AAV1 3’ inverted terminal repeat, an AAV2 3’ inverted terminal repeat, an AAV9 3’ inverted terminal repeat, or a PhP.eB 3’ inverted terminal repeat.

62. The recombinant polynucleotide of claim 49-61, wherein the 5’ inverted terminal repeat is an AAV2 3’ inverted terminal repeat.

63. The recombinant polynucleotide of claim 49-62, wherein the 5’ inverted terminal repeat is a single stranded AAV2 3’ inverted terminal repeat.

64. The recombinant polynucleotide of any one of claims 49-63, wherein the 3 ’ inverted terminal repeat comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 60.

65. The recombinant polynucleotide of any one of claims 1-64, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 3; (ii) SEQ ID NO 10; (iii) SEQ ID NO: 8; (iv) SEQ ID NO: 42; and (v) SEQ ID NO: 64.

66. The recombinant polynucleotide of any one of claims 1-65, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 59; (ii) SEQ ID NO: 3; (iii) SEQ ID NO 10; (iv) SEQ ID NO: 8; (v) SEQ ID NO: 42; (vi) SEQ ID NO: 64; and (vii) SEQ ID NO: 60.

67. The recombinant polynucleotide of any one of claims 1-66, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 3; (ii) SEQ ID NO: 62, wherein the 3’ 33 nucleotides of SEQ ID NO: 3 overlap with the 5’ 33 nucleotides of SEQ ID NO: 62; (iii) SEQ ID NO 10; (iv) SEQ ID NO: 8; (v) SEQ ID NO: 42; and (vi) SEQ ID NO: 64.

68. The recombinant polynucleotide of any one of claims 1-67, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 3; (ii) SEQ ID NO: 66; (iii) SEQ ID NO 10; (iv) SEQ ID NO: 8; (v) SEQ ID NO: 42; and (vi) SEQ ID NO: 64.

69. The recombinant polynucleotide of any one of claims 1-68, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 3; (ii) SEQ ID NO: 56; (iii) SEQ ID NO 10; (iv) SEQ ID NO: 8; (v) SEQ ID NO: 42; and (vi) SEQ ID NO: 64.

70. The recombinant polynucleotide of any one of claims 1-69, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 61 (ii) SEQ ID NO: 3; (iii) SEQ ID NO: 62, wherein the 3’ 33 nucleotides of SEQ ID NO: 3 overlap with the 5’ 33 nucleotides of SEQ ID NO: 62; (iv) SEQ ID NO 10; (v) SEQ ID NO: 8; (vi) SEQ ID NO: 42; and (vii) SEQ ID NO: 64.

71. The recombinant polynucleotide of any one of claims 1-70, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 61 (ii) SEQ ID NO: 3; (iii) SEQ ID NO: 66; (iv) SEQ ID NO 10; (v) SEQ ID NO: 8; (vi) SEQ ID NO: 42; and (vii) SEQ ID NO: 64.

72. The recombinant polynucleotide of any one of claims 1-71, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 61 (ii) SEQ ID NO: 3; (iii) SEQ ID NO: 56; (iv) SEQ ID NO 10; (v) SEQ ID NO: 8; (vi) SEQ ID NO: 42; and (vii) SEQ ID NO: 64.

73. The recombinant polynucleotide of any one of claims 1-72, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 59; (ii) SEQ ID NO: 3; (iii) SEQ ID NO: 62, wherein the 3’ 33 nucleotides of SEQ ID NO: 3 overlap with the 5’ 33 nucleotides of SEQ ID NO: 62; (iv) SEQ ID NO 10; (v) SEQ ID NO: 8; (vi) SEQ ID NO: 42; (vii) SEQ ID NO: 64; and (viii) SEQ ID NO: 60.

74. The recombinant polynucleotide of any one of claims 1-73, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 59; (ii) SEQ ID NO: 3; (iii) SEQ ID NO: 66; (iv) SEQ ID NO 10; (v) SEQ ID NO: 8; (vi) SEQ ID NO: 42; (vii) SEQ ID NO: 64; and (viii) SEQ ID NO: 60.

75. The recombinant polynucleotide of any one of claims 1-74, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 59; (ii) SEQ ID NO: 3; (iii) SEQ ID NO: 56; (iv) SEQ ID NO 10; (v) SEQ ID NO: 8; (vi) SEQ ID NO: 42; (vii) SEQ ID NO: 64; and (viii) SEQ ID NO: 60.

76. The recombinant polynucleotide of any one of claims 1-75, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 59; (ii) SEQ ID NO: 61; (iii) SEQ ID NO: 3; (iv) SEQ ID NO: 62, wherein the 3’ 33 nucleotides of SEQ ID NO: 3 overlap with the 5’ 33 nucleotides of SEQ ID NO: 62; (v) SEQ ID NO 10; (vi) SEQ ID NO: 8; (vii) SEQ ID NO: 42; (viii) SEQ ID NO: 64; and (ix) SEQ ID NO: 60.

77. The recombinant polynucleotide of any one of claims 1-76, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 59; (ii) SEQ ID NO: 61; (iii) SEQ ID NO: 3; (iv) SEQ ID NO: 66; (v) SEQ ID NO 10; (vi) SEQ ID NO: 8; (vii) SEQ ID NO: 42; (viii) SEQ ID NO: 64; and (ix) SEQ ID NO: 60.

78. The recombinant polynucleotide of any one of claims 1-77, wherein the recombinant polynucleotide, from 5’ to 3’, comprises: (i) SEQ ID NO: 59; (ii) SEQ ID NO: 61; (iii) SEQ ID NO: 3; (iv) SEQ ID NO: 56; (v) SEQ ID NO 10; (vi) SEQ ID NO: 8; (vii) SEQ ID NO: 42; (viii) SEQ ID NO: 64; and (ix) SEQ ID NO: 60.

79. The recombinant polynucleotide of any one of claims 1-78, wherein the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 65.

80. The recombinant polynucleotide of any one of claims 1-79, wherein the recombinant polynucleotide comprises a sequence of SEQ ID NO: 65.

81. The recombinant polynucleotide of any one of claims 1-80, wherein the recombinant polynucleotide is SEQ ID NO: 65.

82. The recombinant polynucleotide of any one of claims 1-81, wherein the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 121.

83. The recombinant polynucleotide of any one of claims 1-81, wherein the recombinant polynucleotide comprises a sequence of SEQ ID NO: 121.

84. The recombinant polynucleotide of any one of claims 1-81, wherein the recombinant polynucleotide is SEQ ID NO: 121.

85. The recombinant polynucleotide of any one of claims 1-81, wherein the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 122.

86. The recombinant polynucleotide of any one of claims 1-81, wherein the recombinant polynucleotide comprises a sequence of SEQ ID NO: 122.

87. The recombinant polynucleotide of any one of claims 1-81, wherein the recombinant polynucleotide is SEQ ID NO: 122.

88. The recombinant polynucleotide of any one of claims 1-81, wherein the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 123.

89. The recombinant polynucleotide of any one of claims 1-81, wherein the recombinant polynucleotide comprises a sequence of SEQ ID NO: 123.

90. The recombinant polynucleotide of any one of claims 1-81, wherein the recombinant polynucleotide is SEQ ID NO: 123.

91. The recombinant polynucleotide of any one of claims 1-81, wherein the recombinant polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 124.

92. The recombinant polynucleotide of any one of claims 1-81, wherein the recombinant polynucleotide comprises a sequence of SEQ ID NO: 124.

93. The recombinant polynucleotide of any one of claims 1-81, wherein the recombinant polynucleotide is SEQ ID NO: 124.

94. A plasmid comprising the recombinant polynucleotide of any one of claims 1-93.

95. The plasmid of claim 94, comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 120.

96. The plasmid of claims 94 or 95, wherein the plasmid is SEQ ID NO: 120.

97. A viral vector comprising the recombinant polynucleotide of any one of claims 1-93 or the plasmid of any one of claims 94-96.

98. The viral vector of claim 97, wherein the viral vector is an adenoviral vector, an adeno- associated viral (AAV) vector, or a lentivector.

99. The viral vector of claim 98, wherein the AAV viral vector is selected from the group consisting of AAV 1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV1 1, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-DJ/8, AAV-DJ/9, AAV1/2, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAV.OligoOOl, AAV.SCH9, AAV.r3.45, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PhP.eB, AAV.PhP.Vl, AAV.PHP.B, AAV.PhB.Cl, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, AAVhu68, chimeras thereof, and combinations thereof.

100. The viral vector of claim 98 or claim 99, wherein the AAV vector is an AAV5 vector.

101. The viral vector of any one of claims 98-100, wherein the AAV vector comprises an engineered viral protein (VP) capsid polypeptide.

102. The viral vector of claim 101, wherein the engineered VP capsid polypeptide comprises at least one substitution in a 581-589 region of the engineered VP capsid polypeptide relative to a wild type VP capsid polypeptide of SEQ ID NO: 75.

103. The viral vector of claim 102, wherein the 581-589 region comprises a sequence of SEQ ID NO: 83.

104. The viral vector of claim 102, wherein the 581-589 region comprises a sequence of SEQ ID NO: 84.

105. The viral vector of claim 102, wherein the 581-589 region comprises a sequence of SEQ ID NO: 82.

106. The viral vector of claim 102, wherein the 581-589 region comprises a sequence of SEQ ID NO: 96.

107. The viral vector of claim 102, wherein the 581-589 region comprises a sequence of any one of SEQ ID NO: 85 - SEQ ID NO: 95 or SEQ ID NO: 97 - SEQ ID NO: 111.

108. The viral vector of any one of claims 102-107, wherein the engineered VP capsid polypeptide comprises a formula of (A)-(X)-(B), wherein (A) is a first polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 80, (X) is the 581-589 region, and (B) is a second polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 81.

109. A pharmaceutical composition comprising the recombinant polynucleotide of any one of claims 1-93, the plasmid of any one of claims 94-96, or the viral vector of any one of claims 97- 108, and a pharmaceutically acceptable excipient, carrier, diluent, or combination thereof.

110. A method of expressing a progranulin in a cell, tissue, or subject, the method comprising: delivering a recombinant polynucleotide comprising an RSV promoter and a coding sequence operably linked to the RSV promoter, to the cell, tissue, or subject; and expressing a progranulin encoded by the coding sequence.

111. A method of expressing a progranulin in a cell, tissue, or subject, the method comprising: delivering the recombinant polynucleotide of any one of claims 1-93, the plasmid of any one of claims 94-96, the viral vector of any one of claims 97-108, or the pharmaceutical composition of claim 109 to the cell, tissue, or subject.

112. The method of claim 110 or claim 111, wherein the progranulin is human progranulin.

113. The method of any one of claims 110-112, wherein the progranulin encoded by the coding sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 63.

114. The method of any one of claims 110-113, wherein the progranulin is encoded by a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 10.

115. The method of any one of claims 110-114, wherein the progranulin is encoded by a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 6.

116. The method of any one of claims 110-115, wherein the progranulin is encoded by a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 11.

117. The method of any one of claims 110-116, wherein the progranulin is encoded by a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 26.

118. The method of any one of claims 110-117, wherein the progranulin is encoded by a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 57 and at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to SEQ ID NO: 58.

119. The method of any one of claims 110-118, comprising expressing the progranulin in a fluid secreted by the cell, tissue, or subject.

120. The method of claim 119, wherein the progranulin is expressed in the fluid at a level of not less than 1 ng/mL and not more than 100 ng/mL.

121. The method of claim 119, wherein the progranulin is expressed in the fluid at a level of not less than 10 ng/mL and not more than 100 ng/mL.

122. The method of claim 119, wherein the progranulin is expressed in the fluid at a level of not less than 1 ng/mL and not more than 30 ng/mL.

123. The method of claim 119, wherein the progranulin is expressed in the fluid at a level of not less than 10 ng/mL and not more than 50 ng/mL.

124. The method of claim 119, wherein the progranulin is expressed in the fluid at a level of not less than 10 ng/mL and not more than 30 ng/mL.

125. The method of claim 119, wherein the progranulin is expressed in the fluid at a level of not less than 70 ng/mL and not more than 180 ng/mL.

126. The method of claim 119, wherein the progranulin is expressed in the fluid at a level of not less than 70 ng/mL and not more than 300 ng/mL.

127. The method of any one of claims 119-126, wherein the fluid comprises a cerebrospinal fluid.

128. A method of treating a disease or disorder in a subject in need thereof, the method comprising: delivering a recombinant polynucleotide comprising an RSV promoter and a coding sequence operably linked to the RSV promoter to the subject having frontotemporal dementia; and expressing a progranulin encoded by the coding sequence, thereby treating the subject.

129. A method of treating a disease or disorder in a subject in need thereof, the method comprising: delivering the recombinant polynucleotide of any one of claims 1-93, the plasmid of any one of claims 94-96, the viral vector of any one of claims 97-108, or the pharmaceutical composition of claim 109 to the subject in need thereof, thereby treating the disease or disorder.

130. The method of claim 129 or claim 129, wherein the disease or disorder is frontotemporal dementia, amyotrophic lateral sclerosis (ALS), Alzheimer’s Disease, Parkinson’s Disease, stroke, Gaucher disease, arthritis, limbic-predominant age-related transactivation response DNA-binding protein 43 (TDP-43) encephalopathy, autism, neuronal ceroid lipofuscinosis (e.g., type 11 (CLN11)), dementia, and neurodegeneration; optionally, wherein the neuronal ceroid lipofuscinosis is type 11 ; optionally, wherein neurodegeneration is neurodegeneration associated with normal aging.

131. A method of treating frontotemporal dementia in a subject having frontotemporal dementia, the method comprising: delivering a recombinant polynucleotide comprising an RSV promoter and a coding sequence operably linked to the RSV promoter to the subject having frontotemporal dementia; and expressing a progranulin encoded by the coding sequence, thereby treating the subject.

132. A method of treating frontotemporal dementia in a subject having frontotemporal dementia, the method comprising: delivering the recombinant polynucleotide of any one of claims 1-93, the plasmid of any one of claims 94-96, the viral vector of any one of claims 97-108, or the pharmaceutical composition of claim 109 to the subject having frontotemporal dementia, thereby treating the frontotemporal dementia.

133. The method of any one of claims 128-132, comprising expressing the progranulin in a cerebrospinal fluid of the subject.

134. The method of claim 133, wherein the progranulin is expressed in the cerebrospinal fluid at a level of not less than 10 ng/mL and not more than 100 ng/mL.

135. The method of claim 133, wherein the progranulin is expressed in the cerebrospinal fluid at a level of not less than 1 ng/mL and not more than 100 ng/mL.

136. The method of claim 133, wherein the progranulin is expressed in the cerebrospinal fluid at a level of not less than 10 ng/mL and not more than 30 ng/mL.

137. The method of claim 133, wherein the progranulin is expressed in the cerebrospinal fluid at a level of not less than 1 ng/mL and not more than 30 ng/mL.

138. The method of claim 133, wherein the progranulin is expressed in the cerebrospinal fluid at a level of not less than 10 ng/mL and not more than 50 ng/mL.

139. The method of claim 133, wherein the progranulin is expressed in the cerebrospinal fluid at a level of not less than 10 ng/mL and not more than 30 ng/mL.

140. The method of claim 133, wherein the progranulin is expressed in the cerebrospinal fluid at a level of not less than 70 ng/mL and not more than 180 ng/mL.

141. The method of claim 133, wherein the progranulin is expressed in the cerebrospinal fluid at a level of not less than 70 ng/mL and not more than 300 ng/mL.

142. The method of any one of claims 133-141, wherein an expression level of the progranulin in a serum of the subject is not more than 100-fold, not more than 50-fold, not more than 25-fold, not more than 20-fold, or not more than 10-fold an expression level of the progranulin in the cerebrospinal fluid.

143. The method of any one of claims 133-142, wherein an expression level of the progranulin in a serum of the subject is not less than 0.01 -fold, not less than 0.05 -fold, not less than 0.1-fold, not less than 0.2-fold, not less than 0.25-fold, or not less than 0.5-fold an expression level of the progranulin in the cerebrospinal fluid.

144. The method of any one of claims 133-143, wherein an expression level of the progranulin in a serum of the subject is not more than 300 ng/mL.

145. The method of any one of claims 133-144, wherein an expression level of the progranulin in a serum of the subject is not more than 1500 ng/mL.

146. The method of any one of claims 110-145, wherein the subject is a mammal.

147. The method of claim 146, wherein the mammal is a human, a non-human primate, a rat, a rabbit, a mouse, or a guinea pig.

148. The method of any one of claims 110-147, wherein the delivering comprises intravenous administration.