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Compositions and methods for delivery of cargo to target cells

A technology of goods and delivery systems, applied in the fields of botanical equipment and methods, biochemical equipment and methods, chemical instruments and methods, etc.

Pending Publication Date: 2022-06-10
THE BROAD INST INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, currently used vesicles and particles can be large in size and difficult to generate in a consistent manner

Method used

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  • Compositions and methods for delivery of cargo to target cells
  • Compositions and methods for delivery of cargo to target cells
  • Compositions and methods for delivery of cargo to target cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0319] Expression of various individual env proteins was tested in HEK293T cells ( figure 1). Optimal expression was achieved with Envwl, Envkl and Envfrd (Envw2). The glycoprotein of vesicular stomatitis virus (VSV-G) mediates cell attachment and induces direct fusion between cell membranes. Applicants compared the pseudotyping efficiency of different env proteins with lentiviral DNA. Efficient particle formation was observed with Envk1, Envw1 and Envfrd ( figure 2 ).

[0320] In order to see whether the gag homolog protein Pnma3 is expressed in neuronal cells, applicants fused the gag homolog protein Pnma3 to a red fluorescent reporter protein (RFP) and tested the expression of the gag homolog protein Pnma3 in mouse and rat neurons . The results showed that the expression of this fusion protein was comparable to that of the control RFP-lentiviral construct ( image 3 ).

[0321] Example 2 - Screening of endogenous gag protein candidates for their ability to form caps...

Embodiment 2

[0322] Nine endogenous gag protein candidates were identified and screened for their ability to form vesicles in vitro ( Figure 4 and Figure 5 ). Of the candidates tested, all but Asprv1 were able to form vesicles ( Figure 5 and Table 2). However, only six were able to be secreted from cells (Table 3, Figure 6 ).

[0323] Table 2. Ability of gag protein candidates to form vesicles

[0324]

[0325]

[0326] Table 3. Ability of gag protein candidates to be secreted from cells

[0327] Secreted protein? Asprv1 - Pnma1 + Pnma3 - Pnma4 + Pnma5 + Pnma6 + Pnma7 - Peg10 + Rtl1 +

[0328] Applicants next tested various gag protein candidates for their ability to transfer the Cas9 / gRNA complex to another cell. In the absence of membrane fusion proteins ( Figure 7A ), none of the candidates succeeded in promoting this process. However, including VSV-G ( Figure 7B ) is critical to achieve delivery...

Embodiment 4

[0348] The ability of PEG10 to form vesicles leads to two central questions. 1) How is PEG10 processed, and 2) What is the role of each functional domain? To answer the first question, applicants overexpressed mouse PEG10 with HA tags at the N-terminus and C-terminus in HEK293FT cells, immunoprecipitated PEG10 using HA magnetic beads, and analyzed the bands by Western blotting. The corresponding commassie stained bands were analyzed by mass spectrometry. The results showed that the protein was cleaved into all the respective predicted domains ( Figure 56 , Figures 57A to 57F , Figure 58A and Figure 58B ).

[0349] To answer the second question, applicants compared PEG10 to a previously identified protein called MYEF, a DNA-binding protein that binds a very specific 10 base pair sequence in the 3X repeat (eg Figure 59 shown on the right). Applicants determined that PEG10 binds the exact same sequence, so they attempted to package particles expressing this DNA sequen...

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Abstract

Provided herein are compositions, systems, and methods for delivering cargo to target cells. The compositions, systems, and methods comprise one or more polynucleotides encoding one or more endogenous retroviral elements for forming delivery vesicles and one or more capture moieties for packaging cargo within the delivery vesicles. The one or more endogenous retroviral elements for forming the delivery vesicles may comprise two or more of a retroviral gag protein, a retroviral envelope protein, a retroviral reverse transcriptase, or a combination thereof. The retroviral gag protein alone, the retroviral envelope protein alone, or both the retroviral gag protein and the retroviral envelope protein may be endogenous.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of U.S. Provisional Application No. 62 / 903,127, filed September 20, 2019, and U.S. Provisional Application No. 62 / 003,409, filed April 1, 2020. The entire contents of the above application are hereby fully incorporated by reference. [0003] Statement Regarding Federally Funded Research [0004] This invention was made with Government support under Grant No. HL141201 awarded by the National Institutes of Health. The government has certain rights in this invention. [0005] Electronic Sequence Listing Citation [0006] The contents of the Electronic Sequence Listing ("BROD-4620WP_ST.25.txt", 4,945 bytes in size, created on September 18, 2020) are hereby incorporated by reference in their entirety. technical field [0007] The subject matter disclosed herein generally relates to engineered delivery agents, compositions, systems, and uses thereof. Background technique [0008] The ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/48C12N15/867C07K14/15
CPCC07K14/005C12N15/86C12N2740/14042C12N2740/13022C12N2740/13023C12N2740/14022C12N2740/14023C12N2740/10022C12N2740/10042C12N2740/13042C12N2740/10023C07K14/47C07K2319/735C07K2319/00C12N2310/531C12N2310/20
Inventor F·张M·赛格尔
Owner THE BROAD INST INC
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