Scientists harness human protein to deliver molecular medicines to cells
Made of components found in the human body, the programmable system is a step toward safer, targeted delivery of gene editing and other molecular therapeutics.
By Allessandra DiCorato
Credit: McGovern Institute
SEND packages are introduced to diseased cells to deliver therapeutic mRNA and restore health.
Researchers from MIT, the McGovern Institute for Brain Research at MIT, the Howard Hughes Medical Institute, and the Broad Institute of MIT and Harvard have developed a new way to deliver molecular therapies to cells. The system, called SEND, can be programmed to encapsulate and deliver different RNA cargoes. SEND harnesses natural proteins in the body that form virus-like particles and bind RNA, and it may provoke less of an immune response than other delivery approaches.
The new delivery platform works efficiently in cell models, and, with further development, could open up a new class of delivery methods for a wide range of molecular medicines — including those for gene editing and gene replacement. Existing delivery vehicles for these therapeutics can be inefficient and randomly integrate into the genome of cells, and some can stimulate unwanted immune reactions. SEND has the promise to overcome these limitations, which could open up new opportunities to deploy molecular medicine.
“The biomedical community has been developing powerful molecular therapeutics, but delivering them to cells in a precise and efficient way is challenging,” said CRISPR pioneer Feng Zhang, senior author on the study, core institute member at the Broad Institute, investigator at the McGovern Institute, and the James and Patricia Poitras Professor of Neuroscience at MIT. “SEND has the potential to overcome these challenges.” Zhang is also an investigator at the Howard Hughes Medical Institute and a professor in MIT’s Departments of Brain and Cognitive Sciences and Biological Engineering.
Reporting in Science, the team describes how SEND (Selective Endogenous eNcapsidation for cellular Delivery) takes advantage of molecules made by human cells. At the center of SEND is a protein called PEG10, which normally binds to its own mRNA and forms a spherical protective capsule around it. In their study, the team engineered PEG10 to selectively package and deliver other RNA. The scientists used SEND to deliver the CRISPR-Cas9 gene editing system to mouse and human cells to edit targeted genes.
First author Michael Segel, a postdoctoral researcher in Zhang’s lab, and Blake Lash, second author and a graduate student also in the group, said PEG10 is not unique in its ability to transfer RNA. “That's what’s so exciting,” said Segel. “This study shows that there are probably other RNA transfer systems in the human body that can also be harnessed for therapeutic purposes. It also raises some really fascinating questions about what the natural roles of these proteins might be.”
Inspiration from within
The PEG10 protein exists naturally in humans and is derived from a “retrotransposon” — a virus-like genetic element — that integrated itself into the genome of human ancestors millions of years ago. Over time, PEG10 has been co-opted by the body to become part of the repertoire of proteins important for life.
Four years ago, researchers showed that another retrotransposon-derived protein, ARC, forms virus-like structures and is involved in transferring RNA between cells. Although these studies suggested that it might be possible to engineer retrotransposon proteins as a delivery platform, scientists had not successfully harnessed these proteins to package and deliver specific RNA cargoes in mammalian cells.
Knowing that some retrotransposon-derived proteins are able to bind and package molecular cargo, Zhang’s team turned to these proteins as possible delivery vehicles. They systematically searched through these proteins in the human genome for ones that could form protective capsules. In their initial analysis, the team found 48 human genes encoding proteins that might have that ability. Of these, 19 candidate proteins were present in both mice and humans. In the cell line the team studied, PEG10 stood out as an efficient shuttle; the cells released significantly more PEG10 particles than any other protein tested. The PEG10 particles also mostly contained their own mRNA, suggesting that PEG10 might be able to package specific RNA molecules.
Developing a modular system
To develop the SEND technology, the team identified the molecular sequences, or “signals,” in PEG10’s mRNA that PEG10 recognizes and uses to package its mRNA. The researchers then used these signals to engineer both PEG10 and other RNA cargo so that PEG10 could selectively package those RNAs. Next, the team decorated the PEG10 capsules with additional proteins, called “fusogens,” that are found on the surface of cells and help them fuse together.
By engineering the fusogens on the PEG10 capsules, researchers should be able to target the capsule to a particular kind of cell, tissue, or organ. As a first step towards this goal, the team used two different fusogens, including one found in the human body, to enable delivery of SEND cargo.
“By mixing and matching different components in the SEND system, we believe that it will provide a modular platform for developing therapeutics for different diseases,” said Zhang.
Advancing gene therapy
SEND is composed of proteins that are produced naturally in the body, which means it may not trigger an immune response. If this is demonstrated in further studies, the researchers say SEND could open up opportunities to deliver gene therapies repeatedly with minimal side effects. “The SEND technology will complement viral delivery vectors and lipid nanoparticles to further expand the toolbox of ways to deliver gene and editing therapies to cells,” said Lash.
Next, the team will test SEND in animals and further engineer the system to deliver cargo to a variety of tissues and cells. They will also continue to probe the natural diversity of these systems in the human body to identify other components that can be added to the SEND platform.
“We’re excited to keep pushing this approach forward,” said Zhang. “The realization that we can use PEG10, and most likely other proteins, to engineer a delivery pathway in the human body to package and deliver new RNA and other potential therapies is a really powerful concept.”
This work was made possible with support from the Simons Center for the Social Brain at MIT; National Institutes of Health Intramural Research Program; National Institutes of Health grants 1R01-HG009761 and 1DP1-HL141201; Howard Hughes Medical Institute; Open Philanthropy; G. Harold and Leila Y. Mathers Charitable Foundation; Edward Mallinckrodt, Jr. Foundation; Poitras Center for Psychiatric Disorders Research at MIT; Hock E. Tan and K. Lisa Yang Center for Autism Research at MIT; Yang-Tan Center for Molecular Therapeutics at MIT; Lisa Yang; Phillips family; R. Metcalfe; and J. and P. Poitras.
Produced by the McGovern Institute and Opus Design in collaboration with Feng Zhang, Rhiannon Macrae, and the Broad Institute.
Wataalamu wa sayansi wametumia protini ya binadamu kutoa dawa za molekuli kwa seli, na mfumo huu unaoweza kuandaliwa ni hatua ya kuelekea utoaji salama zaidi wa kuhariri jeni na matibabu mengine ya molekuli. "Kutokana na utafiti uliofanywa na utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti wa utafiti.""" Watafiti kutoka Taasisi ya McGovern ya Utafiti wa Ubongo, Taasisi ya Tiba ya Howard Hughes, na Taasisi ya Broad ya MIT na Harvard wametengeneza njia mpya ya kutoa matibabu ya molekuli kwa seli. Mfumo huo, unaoitwa SEND, unaweza kupangwa ili kuunganisha na kusafirisha mizigo tofauti ya RNA. SEND hutumia protini za asili katika mwili ambazo huunda chembe zinazofanana na virusi na kuunganisha RNA, na inaweza kusababisha majibu ya kinga kidogo kuliko mbinu zingine za utoaji. Jukwaa jipya la utoaji hufanya kazi kwa ufanisi katika mifano ya seli, na, kwa maendeleo zaidi, inaweza kufungua darasa jipya la mbinu za utoaji kwa aina mbalimbali za dawa za molekuli - ikiwa ni pamoja na zile za uhariri wa jeni na uingizwaji wa jeni. Vifaa vya sasa vya matibabu haya vinaweza kuwa na ufanisi na vinaweza kuunganishwa kwa nasibu kwenye genome ya seli, na zingine zinaweza kuchochea athari zisizohitajika za kinga. SEND ina ahadi ya kushinda mapungufu haya, ambayo yanaweza kufungua fursa mpya za kupeleka dawa ya molekuli. "Jumuiya ya biomedical imekuwa kuendeleza nguvu molekuli tiba, lakini kutoa yao kwa seli kwa njia sahihi na ufanisi ni changamoto,"" alisema CRISPR waanzilishi Zhang Feng, mwandishi mwandamizi juu ya utafiti, msingi taasisi mwanachama katika Taasisi Broad, mtafiti katika Taasisi McGovern, na James na Patricia Poitras Profesa wa Neuroscience katika MIT." "Zhang pia ni mtafiti katika Taasisi ya Matibabu ya Howard Hughes na profesa katika Idara za MIT za Sayansi ya Ubongo na Ujuzi na Uhandisi wa Kibiolojia. """ Katika utafiti wao, watafiti walichunguza jinsi SEND (Selective Endogenous Encapsidation for Cellular Delivery) inavyoweza kuunganisha molekuli za binadamu na chembe za binadamu. Katika sehemu ya kati ya SEND kuna protini inayoitwa PEG10, ambayo kwa kawaida huunganisha na mRNA yake mwenyewe na kuunda capsule ya kinga ya mviringo kuzunguka. Katika utafiti wao, timu hiyo ilibuni PEG10 ili kuunganisha na kutoa RNA nyingine kwa njia ya kuchagua. Wataalamu wa sayansi wametumia SEND kuingiza mfumo wa CRISPR-Cas9 wa kuhariri jeni kwa chembe za panya na binadamu ili kuhariri jeni zinazolengwa. Mwandishi wa kwanza Michael Segel, mtafiti wa baada ya udaktari katika maabara ya Zhang, na Blake Lash, mwandishi wa pili na mwanafunzi wa kuhitimu pia katika kundi hilo, walisema PEG10 sio ya kipekee katika uwezo wake wa kuhamisha RNA. "Hiyo ndiyo jambo lenye kusisimua sana", akasema Segel. <unk>Utafiti huu unaonyesha kwamba kuna uwezekano wa mifumo mingine ya uhamisho wa RNA katika mwili wa binadamu ambayo pia inaweza kutumiwa kwa madhumuni ya matibabu. "Hii ni moja ya sababu za msingi za kuonekana kwa ""retrotransposon"" - kiini cha urithi cha virusi - ambacho kimeshiriki katika genome ya mababu za binadamu milioni za miaka iliyopita." Baada ya muda, PEG10 imechukuliwa na mwili kuwa sehemu ya orodha ya protini muhimu kwa maisha. Katika miaka ya hivi karibuni, watafiti wameonyesha kwamba protini nyingine inayotokana na retrotransposon, ARC, huunda muundo unaofanana na virusi na inahusika katika kuhamisha RNA kati ya seli. Ingawa utafiti huo ulionyesha kuwa inaweza kuwa na uwezekano wa kubuni protini za retrotransposon kama jukwaa la utoaji, wanasayansi hawakuwa wamefanikiwa kutumia protini hizi kufunga na kutoa mizigo maalum ya RNA katika seli za mamalia. Kwa kujua kwamba baadhi ya protini zinazotokana na retrotransposon zinaweza kuunganisha na kufunga mizigo ya molekuli, timu ya Zhang iligeukia protini hizi kama magari ya usafirishaji iwezekanavyo. Walitafuta kwa utaratibu katika protini hizo katika chembe za urithi za binadamu ili kupata zile ambazo zingeweza kufanyiza vidonge vya ulinzi. Katika uchunguzi wao wa kwanza, timu hiyo ilipata jeni 48 za binadamu zinazofasiri protini ambazo zinaweza kuwa na uwezo huo. Kati ya hizo, 19 zilikuwa katika panya na wanadamu. Katika mstari wa seli, PEG10 ilijitokeza kama shuttle yenye ufanisi; seli zilitoa chembe nyingi zaidi za PEG10 kuliko protini nyingine yoyote iliyopimwa. PEG10 pia ina mRNA yake mwenyewe, ambayo inaonyesha kuwa inaweza kuwa na uwezo wa kufunga molekuli maalum za RNA. Kwa mfano, katika utafiti wa SEND, wataalamu walipata mfululizo wa molekuli katika mRNA ya PEG10 ambayo PEG10 inatambua na kutumia kuunganisha mRNA yake. Watafiti walitumia ishara hizi kuunda PEG10 na mzigo mwingine wa RNA ili PEG10 iweze kuchagua vifurushi vya RNA. "Kisha, timu hiyo ilifanya vipande vya PEG10 vyenye protini za ziada, zinazoitwa ""fusogens,"" ambazo hupatikana kwenye uso wa seli na kusaidia kuunganisha pamoja." Kwa kuunda fusogens kwenye vidonge vya PEG10, watafiti wanapaswa kuwa na uwezo wa kulenga vidonge kwa aina fulani ya seli, tishu, au kiungo. Katika hatua ya kwanza kuelekea lengo hili, timu ilitumia fusogens mbili tofauti, ikiwa ni pamoja na moja inayopatikana katika mwili wa binadamu, kuwezesha utoaji wa mizigo ya SEND. "Kwa kuchanganya na kulinganisha vipengele tofauti katika mfumo wa SEND, tunaamini kwamba itatoa jukwaa la modular kwa ajili ya kuendeleza matibabu kwa magonjwa tofauti", alisema Zhang. Tiba ya jeni ya SEND inajumuisha protini zinazozalishwa kwa asili katika mwili, ambayo inamaanisha haiwezi kusababisha majibu ya kinga. "Ikiwa utafiti huo utaonesha matokeo mazuri, watafiti wanasema, ""SEND inaweza kufungua fursa za kutoa matibabu ya jeni mara kwa mara na athari ndogo za upande.""" "Teknolojia ya SEND itakamilisha vector za utoaji wa virusi na nanoparticles za lipid kupanua zaidi sanduku la zana za njia za kutoa tiba za jeni na uhariri kwa seli", alisema Lash. Timu hiyo pia imepanga kujaribu SEND kwenye wanyama na kuendeleza mfumo huo ili kuweza kusafirisha bidhaa kwa tishu na seli mbalimbali. Pia wataendelea kuchunguza utofauti wa asili wa mifumo hii katika mwili wa binadamu ili kutambua vipengele vingine vinavyoweza kuongezwa kwenye jukwaa la SEND. "Tunafurahi kuendelea kusukuma mbinu hii mbele", alisema Zhang. "Kutambua kwamba tunaweza kutumia PEG10 na uwezekano mkubwa wa protini nyingine, kubuni njia ya utoaji katika mwili wa binadamu kwa pakiti na kutoa RNA mpya na matibabu mengine ya uwezekano ni dhana yenye nguvu sana. kazi hii ilifanywa iwezekanavyo kwa msaada wa Kituo cha Simons kwa Ubongo wa Jamii katika MIT, Taasisi ya Kitaifa ya Utafiti wa Intramural, Taasisi ya Taifa ya Afya inatoa ruzuku ya 1RHG116117 na 1RHG1111111, Taasisi ya Tiba ya Howard Hughes, Taasisi ya Open Philanthropy, G. Harold na Leila Mathers Charitable Foundation, Edward Mallinckrodt, Jr., Taasisi ya Poitras, Kituo cha Utafiti wa Ugonjwa wa akili katika MIT, na J. J. J. J. Phillips.""" Imetengenezwa na Taasisi ya McGovern na Opus Design kwa kushirikiana na Feng Zhang, Rhiannon Macrae, na Taasisi ya Broad.