Nebraska Lecture with Angie Pannier:

Mike Kamm Author

11/18/2022 Added

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Angie Pannier, Swarts Swarts Family Chair of biological systems engineering, delivered the Nebraska Lecture on Nov. 17, laying out the history of gene therapy and both the challenges and promises ahead.

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[00:00:00.075](upbeat music)
[00:00:03.480]Hello, I'm Chancellor Ronnie Green.
[00:00:06.270]Thank you for joining me for today's Nebraska Lecture.
[00:00:10.020]This distinguished lecture series
[00:00:11.940]features some of the University of Nebraska-Lincoln's
[00:00:14.520]most notable scholars, researchers, artists, and thinkers.
[00:00:19.680]At Nebraska, we believe in the power of every person.
[00:00:23.910]For about two decades,
[00:00:25.530]The Nebraska Lectures have showcased
[00:00:27.390]some of Nebraska's finest scholars,
[00:00:29.640]people who embody the spirit of this institution
[00:00:32.640]and are committed to sharing their knowledge
[00:00:34.860]with the public.
[00:00:36.144](upbeat music)
[00:00:38.760]Our speakers are renowned experts in their fields.
[00:00:41.940]They are scholars who strive to collaborate,
[00:00:44.280]breaking down the barriers between disciplines.
[00:00:47.310]They are educators who are committed to mentoring
[00:00:49.830]and shaping the next generation.
[00:00:52.410]They're problem solvers who have spent their careers
[00:00:55.410]addressing some of society's most pressing challenges.
[00:00:59.310]I am so proud of their accomplishments
[00:01:01.470]and dedication to our university in the state of Nebraska.
[00:01:06.330]Thank you to the Office of Research
[00:01:08.010]and Economic Development,
[00:01:09.810]the university's Research Council,
[00:01:12.030]the Osher Lifelong Learning Institute,
[00:01:14.550]and other partners for making this lecture series possible.
[00:01:18.450]I hope you enjoy today's Nebraska Lecture.
[00:01:27.863](upbeat music)
[00:01:30.840]Good afternoon.
[00:01:31.673]I'm Bob Wilhelm,
[00:01:32.700]vice chancellor for Research and Economic Development
[00:01:35.400]at the University of Nebraska-Lincoln.
[00:01:37.980]Welcome to our fall Nebraska Lecture,
[00:01:40.560]which is part of our annual research days programming.
[00:01:44.310]This lecture series highlights
[00:01:46.050]some of Nebraska's finest artists,
[00:01:48.840]researchers, scholars, and thinkers.
[00:01:51.900]Today's lecture features Dr. Angie Pannier.
[00:01:55.830]Angie's research on such subjects
[00:01:57.990]as non-viral gene delivery, tissue engineering,
[00:02:02.010]DNA vaccination, and stem cell engineering
[00:02:05.580]put her on the cutting edge of some of the most critical
[00:02:09.090]biomedical engineering issues of our time.
[00:02:12.240]Today, she's going to discuss the history of gene therapy,
[00:02:15.720]the events in 2020 that changed the field,
[00:02:19.320]and future technologies
[00:02:20.760]that will revolutionize medical therapies.
[00:02:24.660]Angie received her bachelor's and master's degrees
[00:02:27.720]in biological systems engineering from UNL
[00:02:30.840]and her PhD in biological sciences
[00:02:33.420]from Northwestern University.
[00:02:35.970]She is the Swarts Family Chair
[00:02:38.550]in Biological Systems Engineering.
[00:02:41.400]Angie joined UNL'S faculty in 2007.
[00:02:45.480]Thank you for joining us,
[00:02:46.890]and please stay tuned after the lecture
[00:02:49.410]for a live Q&A with Dr. Pannier.
[00:02:59.370]Well, I'd like to thank Chancellor Ronnie Green
[00:03:01.110]and Vice Chancellor Bob Wilhelm
[00:03:02.400]for the invitation to present
[00:03:03.780]at this prestigious lecture series.
[00:03:05.400]And in particular, also thank my nominator, Dr. Yusong Li,
[00:03:08.850]of the College of Engineering Dean's Office.
[00:03:11.490]I will admit that when I propose the title
[00:03:13.980]DNA and RNA Delivery from Novel Therapies to Vaccines
[00:03:17.220]that End Pandemics quite a few months ago,
[00:03:19.980]I might have been a little bold in my title choice.
[00:03:22.530]And the reason for that is,
[00:03:24.795]one, there is a lot of really fascinating,
[00:03:28.500]interesting, and important science
[00:03:30.450]that has occurred in the last 120 to 150 years
[00:03:33.600]that have led us to where we are today
[00:03:35.610]in terms of novel therapies that make use of DNA and RNA,
[00:03:39.330]and therefore my apologies because I cannot,
[00:03:42.030]for the sake of time, highlight every important scientist
[00:03:45.540]and discovery within this field.
[00:03:47.280]But also I was bold to use the word end
[00:03:49.800]in ending pandemics.
[00:03:52.410]And as we sit here today in November, 2022,
[00:03:55.050]we certainly know that the pandemic has not ended,
[00:03:57.540]but I think that we all would agree
[00:04:00.690]that novel therapies that make use of DNA and RNA
[00:04:04.380]have certainly changed the course of the pandemic.
[00:04:06.720]And I hope over the course of my lecture today
[00:04:08.880]that I explain where we've come, where we're going,
[00:04:11.700]and some of the bright future that I see
[00:04:13.680]ahead in this field.
[00:04:15.930]And so we're gonna start
[00:04:16.763]with talking about biological systems.
[00:04:18.660]And biological systems are made up of organ systems,
[00:04:21.300]and organ systems work coordinatively
[00:04:23.955]to keep us alive and and functioning.
[00:04:26.150]And if we zoom into an organ system,
[00:04:28.320]organ systems are made up of organs.
[00:04:30.000]And we can see on this slide,
[00:04:31.020]I have sort of a nondescript organ there.
[00:04:33.240]Maybe it's a muscle, maybe it's a liver.
[00:04:35.220]But if we zoom into that organ,
[00:04:36.600]we can see that organs are made up of tissue units.
[00:04:39.540]And those tissue units, if we zoom in further,
[00:04:41.670]are made up of individual cells.
[00:04:43.440]And those cells then are diagrammed on the slide,
[00:04:47.250]as diagrammed on the slide,
[00:04:48.810]you can see in that cell diagram
[00:04:50.310]that cells are made up of a variety
[00:04:52.320]of really important features,
[00:04:54.090]some of which I will highlight
[00:04:55.260]that are important to our story today.
[00:04:56.937]And that includes that cells
[00:04:58.650]are enveloped by a plasma membrane.
[00:05:01.230]That's the perimeter or the outside of the cell.
[00:05:03.480]It may not seem like an important part of the cell,
[00:05:05.880]but it controls what enters and leaves the cell.
[00:05:09.180]We also have the cytoplasm within the center of the cell,
[00:05:12.053]and this contains a lot of organelles
[00:05:14.008]and molecular factories
[00:05:15.750]that we'll certainly talk about today as well.
[00:05:18.540]And then we have the nucleus of the cell,
[00:05:20.370]and that really is the heart of our story today.
[00:05:22.770]And in the nucleus, that is where our genes, our DNA,
[00:05:26.280]our genome, resides and controls
[00:05:29.550]not only our traits and our features, but also our diseases.
[00:05:33.630]And we'll talk a little bit about the history
[00:05:35.970]of our genome and our genes
[00:05:37.830]that leads into DNA and RNA technologies
[00:05:40.920]that I wanna describe today.
[00:05:42.570]And so the history starts
[00:05:44.640]with really the father of genetics, Gregor Mendel,
[00:05:47.310]that in the late 1800s,
[00:05:50.123]Gregor Mendel was actually performing
[00:05:52.410]thousands and thousands of experiments with pea plants
[00:05:55.320]where he was crossing pea plants
[00:05:57.060]that had very distinctive features,
[00:05:58.890]including their height, their flower color,
[00:06:01.590]whether their peas were wrinkled or smooth.
[00:06:04.320]And he noticed that when he crossed these plants together,
[00:06:07.438]that he could predict the traits
[00:06:10.170]being passed from parent to child,
[00:06:11.880]or in the case of plants, from generation to generation,
[00:06:15.030]and he started to describe this mathematically
[00:06:17.790]and set up the principles or the fundamentals of heredity.
[00:06:21.900]And in 1909, then Danish botanist Wilhelm Johannsen
[00:06:26.070]first uses the word gene to describe this unit of heredity.
[00:06:30.690]We then move into the early 1900s where Thomas Hunt Morgan
[00:06:34.374]and his group at Columbia University
[00:06:36.480]in their very famous fly room
[00:06:38.850]started working not with plants,
[00:06:40.680]but with fruit flies as their model organism.
[00:06:44.190]They were able to show that these units of heredity,
[00:06:46.650]these genes, as we were starting to call them,
[00:06:49.050]are actually strung on chromosomes.
[00:06:51.300]And these flies are still used today in modern genetics
[00:06:54.960]because of the easy ability to cross these flies
[00:06:58.050]and with features that are easily detectable
[00:07:00.210]or phenotypes is what we would call them.
[00:07:01.890]So different colored eyes or different features of wings.
[00:07:05.280]And it wasn't until 40 years later
[00:07:06.960]that we actually determined
[00:07:08.670]that humans have 46 chromosomes where our genes are arrayed,
[00:07:12.780]and 23 of those chromosomes come from each of your parents.
[00:07:17.160]Working with Dr. Morgan was one of our very own
[00:07:19.525]Nebraska's Nobel Prize winner, George Beadle,
[00:07:22.200]as seen here in an image of his bronze statue
[00:07:25.020]that was recently installed on East Campus.
[00:07:27.420]And George Beadle, along with Edward Tatum,
[00:07:29.850]started using red bread mold as their model organism,
[00:07:33.210]and they showed that the function of this gene,
[00:07:35.580]this unit of her hereditary,
[00:07:37.320]is actually to direct the formation of a particular enzyme,
[00:07:40.530]and importantly, that regulates a chemical event.
[00:07:43.140]And this was really important
[00:07:44.730]in the field of genetics and ecogenetics
[00:07:47.760]where we started to understand
[00:07:49.020]that genes don't just define these outward traits,
[00:07:51.900]but they define the chemical reactions within a cell.
[00:07:56.670]And then famously in 1953, James Watson,
[00:07:59.790]along with Francis Crick,
[00:08:01.020]with data collected by Rosalind Franklin,
[00:08:03.360]characterized the structure of the language of genes,
[00:08:07.800]the DNA double helix.
[00:08:09.570]And here importantly,
[00:08:10.710]they were able to show that that DNA
[00:08:12.900]not only is in the feature of a double helix,
[00:08:16.590]but that the backbone
[00:08:18.300]is made up of sugars and phosphate groups,
[00:08:20.250]and these run in two strands
[00:08:22.560]that are actually anti-parallel to each other.
[00:08:24.510]And the ladders of this helix are made up of bases
[00:08:27.570]that pair in a very specific way
[00:08:29.520]and that form a sequence of an alphabet, if you will,
[00:08:33.900]that helps to write the code of genes.
[00:08:37.830]And so fast forwarding many years and decades worth of work,
[00:08:42.330]this is our understanding of DNA and the DNA code.
[00:08:45.390]And so we know that we have chromosomes,
[00:08:47.160]genes are arrayed on chromosomes,
[00:08:49.230]and these chromosomes are found in the nucleus of cells.
[00:08:52.050]If we take that chromosome and we unravel it,
[00:08:54.150]we see that characteristic double helix structure
[00:08:56.790]of the DNA molecule.
[00:08:58.140]And we see that those rungs of those ladders
[00:09:00.450]are made up of what we call bases
[00:09:03.120]that one is attached to one strand,
[00:09:05.130]another base is attached to the other strand,
[00:09:06.990]and they pair as a stair step
[00:09:09.780]together in a very specific way.
[00:09:11.790]And that A always pairs with T and G always pairs with C.
[00:09:15.390]And not only is that important
[00:09:17.400]to the structure and function of DNA,
[00:09:20.070]it allows for DNA to replicate when cells divide,
[00:09:22.950]it allows for DNA to be read as a code,
[00:09:26.550]but also the sequence of letters are important,
[00:09:29.653]and that's the way that genetic information is encoded
[00:09:34.050]in a cell in an organism.
[00:09:36.690]And so this really leads us to the central dogma of biology.
[00:09:40.410]And that is the idea
[00:09:41.730]that if we remember back to our cell diagram,
[00:09:43.980]and I've now simplified it to a cell with a membrane,
[00:09:47.250]a cytoplasm and a nucleus,
[00:09:49.140]within the nucleus is where these chromosomes are,
[00:09:51.780]where these highly bundled areas of DNA are.
[00:09:54.960]I always tell my students to picture chromosomes
[00:09:57.090]like uncooked ramen noodles.
[00:09:58.830]That's how highly packed the DNA is in the chromosomes.
[00:10:01.740]And these chromosomes are like books,
[00:10:03.900]books of instructions,
[00:10:05.460]and they have on them genes,
[00:10:07.650]which are like book chapters
[00:10:09.150]that are instructions for very specific proteins
[00:10:12.510]or chemical reactions as George Beadle described them,
[00:10:15.510]that actually carry out the function of the cell,
[00:10:17.790]and this happens every second in every cell of your body.
[00:10:21.300]And so biology is really smart
[00:10:23.430]in that we have to protect this genetic code,
[00:10:26.130]these book chapters, these genes,
[00:10:27.781]that are housed on these chromosomes,
[00:10:29.730]so we keep them safely locked away within the nucleus,
[00:10:32.580]which in itself, has its own membrane.
[00:10:35.280]And instead we decide that certain cells
[00:10:37.380]only need to read certain instructions
[00:10:39.656]or use certain book chapters or express certain genes
[00:10:44.370]given what the cell needs to do
[00:10:46.020]or the functions it needs to perform.
[00:10:47.940]And so what happens is,
[00:10:49.110]there's cell machinery within the nucleus
[00:10:51.210]that reads the code,
[00:10:52.410]the sequence of those letters in the DNA,
[00:10:55.500]and it transcribes it into the language of messenger RNA.
[00:10:58.740]Now, messenger RNA looks very similar in language to DNA.
[00:11:02.310]However, it is single stranded,
[00:11:04.240]and it is immediately shuttled
[00:11:06.300]into the cytoplasm of the cell
[00:11:08.430]where that recipe for a protein
[00:11:10.890]can immediately be acted upon
[00:11:12.810]by other molecular factors called ribosomes
[00:11:15.480]that actually translate the code of messenger RNA
[00:11:18.810]into the language of amino acids,
[00:11:21.120]which are the subunits that string together
[00:11:23.520]to form a protein.
[00:11:24.570]And so this is read in triplets of bases called codons,
[00:11:29.220]and then translated into a protein.
[00:11:31.590]And the protein is what carries out
[00:11:33.300]the function of your cell.
[00:11:34.440]And so a nerve cell or an intestinal cell,
[00:11:37.260]they are that type of cell because of the proteins
[00:11:40.020]that they are expressing,
[00:11:41.070]because of the book chapters or the genes
[00:11:43.470]that they're reading in the nucleus.
[00:11:45.510]And so you can imagine knowing this information,
[00:11:48.600]it became very attractive
[00:11:50.340]that what if we delivered DNA to the nucleus of a cell?
[00:11:55.110]What if we delivered a gene to the nucleus of the cell?
[00:11:57.690]We could put our own instructions in
[00:12:00.330]that the cell would then use in the same way
[00:12:02.850]it's using the instructions on the chromosome.
[00:12:05.460]And so early on, scientists started to see
[00:12:08.220]if we could do this.
[00:12:09.180]Could we deliver DNA to cells?
[00:12:11.940]And in 1961, Professor Lorraine Kraus
[00:12:14.820]at the University of Tennessee delivered DNA
[00:12:17.400]that encoded for hemoglobin, a protein,
[00:12:19.920]from a healthy patient cell and took that DNA
[00:12:23.010]and delivered it to cells from the bone marrow
[00:12:25.110]of a patient with sickle cell anemia,
[00:12:27.090]and was able to show that in the cells
[00:12:29.640]from the patient with sickle cell anemia,
[00:12:31.740]they were able to not only have that DNA, that book chapter,
[00:12:35.100]that instruction for the healthy patient hemoglobin
[00:12:38.220]in the nucleus,
[00:12:39.420]but those cells read those instructions
[00:12:41.340]and produced a functional hemoglobin.
[00:12:44.010]Now, her techniques were very rudimentary
[00:12:46.890]and it wasn't very efficient
[00:12:48.390]in the way that she attempted to deliver DNA to cells,
[00:12:51.390]but it did show that we could put instructions in our cells.
[00:12:56.310]But we decided we needed to find a new
[00:12:58.170]and more effective way to deliver genes to cells.
[00:13:01.050]And it turns out that in many cases,
[00:13:03.180]nature already has determined a way to do many of the things
[00:13:06.300]that we as biomedical engineers and scientists
[00:13:09.314]are trying to do.
[00:13:10.620]And in this case, we look to viruses.
[00:13:13.230]And so this is a generic description of a virus
[00:13:16.440]and, really, a virus life cycle.
[00:13:18.000]And you can see that the virus
[00:13:19.800]is made up of a genome or genes that it carries,
[00:13:25.110]and then it has sort of a capsule or an outer coating.
[00:13:27.780]And a virus is an obligate parasite
[00:13:30.094]and that it cannot replicate on its own.
[00:13:32.730]So in a normal infection,
[00:13:34.334]what happens is the virus enters a cell,
[00:13:37.050]it delivers its genes, its instructions,
[00:13:39.764]its book chapters to the nucleus of the host cell
[00:13:43.920]to instruct that cell to make more copies of the virus,
[00:13:46.920]and then the virus buds off more copies.
[00:13:49.080]And so we started to think as scientists,
[00:13:51.300]well, this is an example of nature delivering genes.
[00:13:54.180]So what if we could use viruses to do this,
[00:13:56.910]but to deliver the gene that we want to deliver?
[00:13:59.970]And so in really outstanding work
[00:14:03.185]by Rogers and Pfuderer in 1968,
[00:14:07.260]they published in Nature
[00:14:08.820]that they were able to take a tobacco mosaic virus
[00:14:12.189]and insert a gene of their choice into that virus,
[00:14:16.020]and then use that engineered virus
[00:14:18.180]to deliver that gene to tobacco plants or tobacco leaves.
[00:14:21.210]And they were able to demonstrate
[00:14:22.590]that they were able to accomplish the gene delivery.
[00:14:26.880]And so this set off a lot of excitement
[00:14:29.280]both in the areas of delivering genes to plants,
[00:14:31.800]which is certainly set up a huge field
[00:14:33.660]of genetically modified organisms,
[00:14:35.520]as well as the ability to use tobacco plants
[00:14:38.370]to produce pharmaceuticals,
[00:14:40.110]but it also, within the mammalian cell world,
[00:14:42.360]started to to help us see
[00:14:44.130]how we might be able to deliver genes to mammalian cells.
[00:14:48.360]Again, not using a tobacco mosaic virus,
[00:14:50.700]but using viruses that we know could infect human cells.
[00:14:55.260]And so the concept of gene therapy
[00:14:58.260]started to become just amazingly exciting
[00:15:02.730]because at this point,
[00:15:04.380]we have just demonstrated that genes
[00:15:06.690]instruct the formation of proteins,
[00:15:08.880]we have shown that viruses can deliver genes,
[00:15:12.120]and we have started to get information that at the time,
[00:15:16.200]we were able to link over 1,500 diseases
[00:15:18.690]to some sort of genetic bases.
[00:15:21.000]And in fact, of those 92 of the diseases,
[00:15:24.120]we knew which gene was causing the disease,
[00:15:27.330]which means that there was some mutation in the DNA code
[00:15:31.209]that was causing the protein
[00:15:32.953]that was expressed to be incorrect in some way
[00:15:36.360]and causing the disease.
[00:15:37.590]And so this seems an amazing point in time
[00:15:41.111]when things started to converge.
[00:15:42.990]The ability to deliver genes
[00:15:44.670]and the ability to know which genes to deliver.
[00:15:46.980]And so Freedman and Roblin
[00:15:48.300]outlined the idea of gene therapy,
[00:15:50.569]and this is their paper from 1972
[00:15:55.530]where they described could we use genes
[00:15:58.620]as therapy in this idea of gene therapy?
[00:16:00.657]And I have to say to all the students,
[00:16:03.240]or anyone that is any field,
[00:16:04.770]it's really exciting to go back and read the paper
[00:16:06.990]that set forth your field,
[00:16:09.240]because not only do they describe the immense potential
[00:16:13.290]of using genes as a therapy
[00:16:15.720]and really outline almost everything that we have
[00:16:19.080]and are continuing to work on
[00:16:20.640]and and saw all of that in 1972,
[00:16:24.030]but they also urged caution
[00:16:25.680]and precaution that we probably should have heated
[00:16:28.430]a little stronger than we did,
[00:16:29.940]and that will come clear here in a moment,
[00:16:32.820]'cause they said this could be dangerous.
[00:16:34.560]This could be scary if not done correctly.
[00:16:37.200]And so this paper is just a fantastic example
[00:16:40.168]of two wonderful scientists forming a field,
[00:16:44.370]giving the parameters, and setting through the warning.
[00:16:46.980]They also have the most exquisitely simplistic,
[00:16:49.537]beautiful figure within this paper.
[00:16:51.930]And that figure says,
[00:16:53.550]we have to figure out how to deliver these genes.
[00:16:55.830]And that is we have to take a gene
[00:16:57.450]and get it across the plasma membrane through the cytoplasm
[00:17:01.470]and across the nuclear envelope
[00:17:03.150]to actually reside in the nucleus of the cell
[00:17:05.310]we're trying to deliver the gene to.
[00:17:06.810]And they show that one way to do that might be a virus.
[00:17:09.510]And we started to demonstrate in the field that viruses
[00:17:11.730]could be re-engineered to do that,
[00:17:13.530]but they also said maybe there's just another way
[00:17:15.630]to just get DNA itself into the nucleus.
[00:17:19.230]And so at the time there was individuals
[00:17:21.090]trying to use things like calcium phosphate,
[00:17:23.760]trying to use some early types of polymers
[00:17:26.460]to accomplish this.
[00:17:28.020]But in 1987, Felgner and colleagues
[00:17:30.090]report the synthesis of a cationic lipid.
[00:17:32.700]This is a highly, positively charged lipid
[00:17:35.340]that could interact with DNA.
[00:17:36.930]And in this case, the DNA is actually a circular piece
[00:17:40.290]of DNA called plasma DNA,
[00:17:42.240]and that plasma DNA includes the gene
[00:17:44.670]you're trying to deliver.
[00:17:45.780]And they complex this cationic lipid with this DNA
[00:17:49.830]to form a complex or a nanoparticle
[00:17:52.023]that they were able to show can deliver DNA into cells
[00:17:57.780]and into a variety of cells.
[00:17:59.520]And so this starts to suggest
[00:18:00.810]that maybe we don't have to use a virus.
[00:18:02.610]There may be other modalities or methods that we could use.
[00:18:06.150]And it really starts to set up sort of two areas
[00:18:08.760]within the RNA and DNA delivery world,
[00:18:11.250]and that's viral gene delivery and non-viral gene delivery.
[00:18:14.280]And full disclosure,
[00:18:15.270]I work in the area of non-viral gene delivery,
[00:18:17.610]and I'll certainly come back to that
[00:18:18.930]at the end of the lecture today.
[00:18:20.490]But these two areas worked quickly
[00:18:24.856]and in ways that were very innovative
[00:18:28.140]to try to develop both viral and non-viral techniques
[00:18:30.851]to deliver genes.
[00:18:32.340]So the viral community worked on identifying viruses
[00:18:35.091]that are known to infect human cells,
[00:18:37.860]and that we would be able to engineer out the parts
[00:18:40.710]that cause infection or the virus to replicate
[00:18:43.216]and just retain the parts that deliver the gene.
[00:18:46.200]And the non-viral gene delivery community
[00:18:48.180]started to come up with more lipids and more polymers
[00:18:50.730]and more materials to effectively complex the DNA
[00:18:54.570]and deliver it to a cell.
[00:18:56.220]Now, the viral gene delivery community
[00:18:59.040]started to make much larger strides
[00:19:01.260]than the non-viral gene delivery community.
[00:19:03.030]And for one reason, viral vectors,
[00:19:05.610]which is the term we use for this,
[00:19:07.110]were based on many decades of virology work
[00:19:11.010]and millions of years of evolution
[00:19:13.650]that viruses were intended to deliver genes.
[00:19:17.718]And so because of that,
[00:19:19.078]the viral vector started to make some big splashes.
[00:19:22.260]And in 1990,
[00:19:23.880]The New England Journal of Medicine
[00:19:25.230]was the first report of an officially approved demonstration
[00:19:28.140]of gene therapy in humans.
[00:19:30.000]And what Rosenberg and Anderson did
[00:19:31.710]was they removed tumor infiltrating lymphocytes
[00:19:35.190]from a patient with a terminal malignant melanoma,
[00:19:38.400]they used a virus to deliver DNA to those cells,
[00:19:41.700]and then they reinfused the patient's cells back
[00:19:44.070]into the same patient.
[00:19:45.210]And all of the patients in this study,
[00:19:46.980]there was five, with terminal malignant melanoma,
[00:19:49.995]they were able to show in all of these patients
[00:19:52.170]that they had the cells with the delivered genes,
[00:19:54.420]there was no ill effects,
[00:19:55.650]and some of the patients responded positively to treatment.
[00:19:59.160]But the most important part of this study
[00:20:01.080]was that it established the feasibility
[00:20:03.480]and the idea of removing cells, delivering genes,
[00:20:06.090]and then giving them back to the patient,
[00:20:07.500]and the safety of gene therapy.
[00:20:09.180]This was an exciting time.
[00:20:10.530]So these ideas,
[00:20:11.640]these this hundred years of molecular biology
[00:20:13.890]combined with innovations in viral vectors
[00:20:16.500]was starting to come to fruition.
[00:20:19.290]Also at the same time, the NIH,
[00:20:22.290]along with other agencies launched the Human Genome Project.
[00:20:25.860]And this happened at this moment in time
[00:20:28.650]because the technology that was needed
[00:20:30.810]to sequence large amounts of DNA
[00:20:32.580]started to really be available.
[00:20:36.629]Nowhere near as fast or as accurate
[00:20:40.259]as the technology we have today,
[00:20:42.750]but it did allow for large scale sequencing.
[00:20:45.677]And so the goal of this project
[00:20:47.490]was to identify every gene in the human
[00:20:50.280]and to understand the function of that gene,
[00:20:52.290]also to understand its potential implications in diseases,
[00:20:56.280]to be able to use that information to cure diseases.
[00:20:58.830]And these sequences were uploaded in real time.
[00:21:01.170]And so now not only do we have the way to deliver genes,
[00:21:03.866]and we've shown safety in humans,
[00:21:06.420]but we have all this data coming out
[00:21:09.330]that suggests other targets, other genes,
[00:21:11.460]that we might be able to deliver.
[00:21:12.780]So a very exciting time.
[00:21:15.060]So much hope and so much enthusiasm
[00:21:17.310]in the idea of using genes as medicines.
[00:21:21.060]And indeed, in 1990,
[00:21:24.240]we then started a clinical trial of the idea
[00:21:27.930]of actually delivering a functional gene to a human,
[00:21:31.380]and it was the field's first success.
[00:21:34.110]So in 1990, four-year-old Ashanthi de Silva
[00:21:36.870]was one of two children
[00:21:38.070]in the first successful gene therapy clinical trial.
[00:21:40.890]And so she was born
[00:21:41.970]with a severe combined immunodeficiency or SCID,
[00:21:44.754]and she was deficient or lacked
[00:21:48.210]the enzyme adenosine deaminase.
[00:21:50.340]And without this enzyme,
[00:21:51.930]her T-cells died and she was unable to fight infections.
[00:21:54.570]This is bubble boy syndrome.
[00:21:56.190]So the idea that you couldn't leave your house,
[00:21:58.710]you couldn't even get a simple cold
[00:22:00.570]because it could be fatal.
[00:22:02.550]And so what researchers did to Ashanthi
[00:22:05.430]as well as the other child in the clinical trial
[00:22:07.860]was they removed some of her blood cells,
[00:22:10.230]they delivered this ADA or this gene
[00:22:12.780]that encoded for this enzyme to her cells,
[00:22:14.880]and they readministered her cells to her
[00:22:17.280]over a course of two years.
[00:22:19.110]And again, this was done in 1990.
[00:22:21.330]This is an image of Ashanthi from two years ago.
[00:22:24.060]She's still alive today,
[00:22:25.980]and this was huge celebration, right?
[00:22:28.860]This showed us that, again, the idea
[00:22:31.474]not only that we can safely deliver genes,
[00:22:34.350]but that we can maybe cure someone,
[00:22:37.230]and that's a very strong word to use.
[00:22:39.960]But it's not intended to be a treatment,
[00:22:42.240]it's intended to replace a gene
[00:22:44.130]that has mutated and be a cure.
[00:22:46.740]However, in the late 1990s,
[00:22:48.448]we had devastating setbacks,
[00:22:50.730]and devastating probably is too light of a word to use.
[00:22:54.270]But in 1999, 18-year-old Jesse Gelsinger,
[00:22:56.970]a name that is now known to every person
[00:22:59.760]that works in gene therapy throughout the entire world,
[00:23:02.160]had a genetic condition
[00:23:03.270]known as ornithine transcarbamylase deficiency,
[00:23:06.870]which basically limited the ability of his liver
[00:23:10.650]to break down toxic ammonia, which accumulated in his blood.
[00:23:13.950]And this led to...
[00:23:15.540]He was able to manage the disease
[00:23:17.220]with diet and many, many different medications
[00:23:19.560]taken every day,
[00:23:20.430]but he entered a clinical trial where the idea was that,
[00:23:24.090]a working copy of the gene that encoded that enzyme
[00:23:27.022]that was meant to break down ammonia
[00:23:28.890]was delivered to his liver cells using an adenovirus.
[00:23:32.790]This is a virus that causes the common cold in humans.
[00:23:36.510]However, four days after being treated,
[00:23:38.280]Gelsinger died after suffering a fatal immune reaction
[00:23:41.580]to the treatment.
[00:23:42.413]In fact, it was determined,
[00:23:43.350]it was a reaction to the adenovirus.
[00:23:45.840]So the viral vector that was delivering the gene.
[00:23:50.070]And after that, there was another clinical trial
[00:23:53.340]of patients being treated again for SCID
[00:23:56.040]where 5 of the 20 patients,
[00:23:57.690]so a significant number,
[00:23:58.860]went on to develop leukemia
[00:24:00.480]because of the virus used in that treatment.
[00:24:03.000]And so the field had to take a very long pause
[00:24:07.350]and revisit and rework our delivery methods
[00:24:11.220]and really rethink through
[00:24:13.140]how can we even more safely engineer these viral vectors?
[00:24:17.130]How can we design new materials
[00:24:19.170]in the non-viral gene delivery space?
[00:24:21.060]And I will admit, in the early 2000s,
[00:24:23.580]I think a lot of us thought
[00:24:24.810]that our road back was going to take a very long time.
[00:24:28.140]The FDA was extremely careful
[00:24:31.440]and reexamined their approach to clinical trials.
[00:24:34.020]And it was a good pause.
[00:24:35.640]It was a pause that we all needed,
[00:24:37.474]but was led to that pause
[00:24:39.720]because of some really unfortunate outcomes.
[00:24:42.840]But after working through details of clinical trials,
[00:24:45.690]after working through re-engineering safer vectors,
[00:24:49.290]we started to see some successes.
[00:24:51.150]There was one drug that was approved in China
[00:24:53.910]and one drug approved in in Russia.
[00:24:56.242]But in 2012, Glybera was market approved
[00:25:01.063]as the first gene therapy approved for use
[00:25:03.750]in Western countries.
[00:25:04.920]And it was approved by the European Medicines Agency.
[00:25:07.920]It was never approved by the USFDA.
[00:25:10.350]And it was using an adenovirus.
[00:25:11.730]So that same virus that had been used in Jesse's trial,
[00:25:14.580]but now re-engineered, rethought through to be much safer.
[00:25:19.320]And the idea was to deliver a gene
[00:25:20.970]to reverse lipoprotein lipase deficiency,
[00:25:23.200]which was a rare genetic disorder.
[00:25:27.150]It was herald it is a huge success.
[00:25:29.520]We had our first gene therapy product.
[00:25:32.220]However, it was priced at $1 million
[00:25:35.280]and was withdrawn from the market after only five years
[00:25:38.430]after only 31 patients received it,
[00:25:40.860]and 30 of those patients received it on clinical trial.
[00:25:43.530]So only one person in the entire world
[00:25:45.360]was ever prescribed this treatment.
[00:25:47.040]And so while it showed again
[00:25:49.890]that we had overcome some of our challenges with safety,
[00:25:54.140]it it highlighted new challenges that it's extremely hard
[00:25:57.930]to manufacture safely these medicines
[00:26:01.953]that if we have a very rare target disease,
[00:26:05.430]there's a small patient population
[00:26:07.209]to recover R&D costs or cost of manufacturing.
[00:26:11.610]And this also demonstrated that the FDA approval
[00:26:14.288]was a rough road.
[00:26:16.560]And so things sort of marched slowly again.
[00:26:21.180]But around five years later,
[00:26:23.040]that's when the US market started to really open up
[00:26:25.620]to gene therapy products.
[00:26:27.060]And beginning in 2017,
[00:26:28.950]the FDA approved the CAR T therapies,
[00:26:31.410]and this is a gene therapy for blood cancer.
[00:26:34.200]And this is similar to some of the earlier studies
[00:26:36.480]with the idea that we would take a patient's cells,
[00:26:38.790]deliver genes, and then give those cells back
[00:26:40.800]to the same patient.
[00:26:41.850]And so in this procedure, a patient that presents
[00:26:45.780]with a certain type of blood cancer,
[00:26:47.964]there is a biopsy taken and blood cells are collected
[00:26:50.823]and sorted for T-cell.
[00:26:52.500]These are a specific type of immune cell.
[00:26:55.020]These T-cells then are used,
[00:26:57.180]and we use a virus to deliver a gene to these T-cells
[00:27:00.570]called a chimeric antigen receptor.
[00:27:02.340]And this is actually not a gene that is missing or mutated,
[00:27:05.940]it's actually a new gene that allows these cells to,
[00:27:08.880]when they read the code of the gene
[00:27:10.620]and express the protein encoded in it,
[00:27:12.810]they present a receptor or a CAR
[00:27:15.030]on the outside of their surface.
[00:27:17.340]We then grow up lots of these cells
[00:27:19.380]that have this receptor on their surface,
[00:27:21.150]re-administer it back to the patient,
[00:27:22.950]and those receptors are like a homing beacon
[00:27:25.440]that allows them to find cancer cells and kill them.
[00:27:28.290]And now, since 2017,
[00:27:30.720]we now have six different CAR T therapies
[00:27:32.970]that are approved in the US by the FDA
[00:27:35.400]to treat various forms of leukemia and lymphoma.
[00:27:39.270]And in some cases, these represent nearly a cure
[00:27:42.840]for these cancer patients.
[00:27:46.170]We have other therapies
[00:27:47.520]that are changing lives, FDA approved.
[00:27:50.040]So in 2017, the FDA also approved Luxturna,
[00:27:52.830]which was the first gene therapy
[00:27:54.360]that treats an inherited condition for blindness.
[00:27:57.060]And so this is a form of blindness
[00:27:59.672]that is caused by a mutation in a gene called RPE65.
[00:28:03.810]And if a patient presents with that,
[00:28:06.000]we can use this therapy
[00:28:08.197]that uses an adeno-associated viral vector,
[00:28:10.626]so like an adenovirus,
[00:28:12.343]but considered to be a little safer,
[00:28:15.510]to deliver a functional copy of that gene
[00:28:17.793]into the retinal pigment epithelial cells of the eye,
[00:28:20.640]and these patients go from being blind to being able to see.
[00:28:24.330]If you go watch any of the videos online about this,
[00:28:26.880]it is just completely filled with hope and amazement.
[00:28:32.520]Like other therapies,
[00:28:33.840]this therapy costs around 400,000 per eye treated.
[00:28:39.420]Just this year, the FDA has now approved
[00:28:41.490]two other new gene therapies,
[00:28:43.560]a gene therapy for beta thalassemia treatment,
[00:28:46.530]and Zolgensma, which treats spinal muscular atrophy
[00:28:50.100]in infants.
[00:28:52.260]And it should be noted that Zolgensma
[00:28:55.200]is a one-time therapy to cure these individuals,
[00:28:58.410]and right now priced at 2.125 million per dose.
[00:29:02.160]It is considered to be the most expensive medicine
[00:29:04.230]in the world.
[00:29:07.110]Finally, in just this year,
[00:29:09.362]the European Commission
[00:29:10.770]granted conditional marketing authorization to Rictavian,
[00:29:14.100]which is a gene therapy
[00:29:15.420]for the treatment of severe hemophilia A.
[00:29:17.623]A hemophilia is a disease
[00:29:19.050]that affects a large patient population
[00:29:21.390]with various severe outcomes.
[00:29:22.830]And this marks really a target
[00:29:25.230]that has been, for gene therapists,
[00:29:27.030]something they've been looking for for a long time.
[00:29:28.830]Hemophilia is characteristically caused
[00:29:30.510]by mutation in a gene,
[00:29:31.740]and we've been trying for a long time
[00:29:33.420]to deliver a cure in that way.
[00:29:35.220]And so while I've talked about applications,
[00:29:40.050]and I'll certainly come back to comment more about the cost
[00:29:42.600]because cost is an issue in this field,
[00:29:45.751]what I've described so far
[00:29:47.700]is really what's called in vivo and ex vivo gene therapy.
[00:29:50.318]The idea that in vivo means in our patient,
[00:29:53.310]ex vivo means cells that we take out of our patient,
[00:29:56.130]we deliver a gene, and give back to our patient.
[00:29:58.621]I've described the ideas
[00:30:00.480]of using those kinds of gene therapies
[00:30:02.400]to deliver a gene to be, quote, unquote,
[00:30:06.217]"a cure for a disease."
[00:30:07.710]But there's other applications
[00:30:08.970]where you might wanna deliver a gene,
[00:30:10.560]and that includes things like genome editing,
[00:30:13.320]which I'll talk briefly about here at the end,
[00:30:15.420]or tissue engineering,
[00:30:16.590]using gene therapy to drive regeneration of tissue
[00:30:19.070]for replacement, but also vaccination.
[00:30:21.990]And this timing and the long history we have
[00:30:25.710]within the field of also looking at vaccination
[00:30:27.860]as an application for gene therapy,
[00:30:30.300]the the timing of that was really pretty perfect.
[00:30:33.090]If we look back two and a half years,
[00:30:35.040]almost three years ago,
[00:30:36.270]in January, 2020 when we all started to see
[00:30:38.550]these new reports of a pneumonia like illness in China,
[00:30:43.530]and then we saw the first case of SARS-CoV-2 or COVID-19
[00:30:48.150]in the US in late January, 2020.
[00:30:51.870]And so as individuals within this field
[00:30:55.320]that are working on DNA delivery and RNA delivery,
[00:30:58.680]which I haven't talked much about,
[00:30:59.850]but we'll hear in a moment,
[00:31:01.380]I think we saw this was our chance, right?
[00:31:03.750]We knew that as this pandemic began,
[00:31:06.346]that we were gonna need a vaccine approach
[00:31:09.330]that was gonna allow for fast manufacture,
[00:31:12.900]very efficient, efficacious outcomes,
[00:31:15.870]and the ability to mobilize it quickly and across the globe.
[00:31:19.350]And so if we look at sort of traditional approaches
[00:31:21.960]to vaccine, which are on the left side of the screen,
[00:31:24.690]what we typically do
[00:31:25.650]is that we actually have to get the virus
[00:31:27.390]that we're trying to vaccinate against,
[00:31:28.980]and then inactivate it in some way,
[00:31:31.230]and re-administer it back to our patients.
[00:31:33.210]And this can take time.
[00:31:34.140]It can take time to isolate the virus,
[00:31:36.120]it can definitely take time
[00:31:37.080]to grow up massive amounts of it, purify it,
[00:31:39.027]and this is why it takes us months
[00:31:40.740]to get ready for an influenza vaccine, for instance.
[00:31:43.770]But there are other ways to accomplish that,
[00:31:45.930]including using DNA or RNA,
[00:31:48.570]and delivering it with non-viral means
[00:31:51.030]or delivering it with viral vectors.
[00:31:52.830]Not the virus that's causing the infection,
[00:31:55.560]but other viruses that we know can deliver genes.
[00:31:58.800]And so that's where the field really settled quite quickly
[00:32:02.250]because we knew
[00:32:03.083]that we had decades worth of research in this area
[00:32:05.940]and we knew that these were technologies
[00:32:07.500]that would allow us to mobilize quickly.
[00:32:09.510]And so I wanna remind us of the central dogma of biology.
[00:32:12.570]If we remember that our DNA is in our nucleus,
[00:32:15.690]it's transcribed into messenger RNA
[00:32:17.910]that's shuttled into the cytoplasm,
[00:32:19.650]and then translated into a protein,
[00:32:21.870]everything we've been talking about today
[00:32:23.340]is to really convince a cell or to deliver a gene to a cell
[00:32:26.370]to correct a mutation.
[00:32:27.900]In the case of a vaccine,
[00:32:29.490]the idea is to deliver a gene to the cell
[00:32:31.980]that the cell wouldn't normally express,
[00:32:34.200]but then a virus would.
[00:32:36.030]And that, in turn, can stimulate an immune response
[00:32:39.600]without ever actually using the entire virus in the vaccine.
[00:32:43.320]Only one gene.
[00:32:44.730]One piece of DNA that encodes for a part of the virus.
[00:32:48.720]And so we knew for the coronavirus,
[00:32:51.810]and the structure is shown in the left,
[00:32:53.910]that it has a genome like all viruses,
[00:32:56.430]and on its surface, it has this spike protein.
[00:32:59.580]And if you look at the coronavirus in microscopy images,
[00:33:02.790]it looks like a corona,
[00:33:03.960]which is why it's called a coronavirus.
[00:33:05.700]Well, we knew because of the MERS
[00:33:07.260]and the SARS outbreaks of the last 20 years
[00:33:09.600]that that spike protein is a good target for a vaccine.
[00:33:13.260]And so the idea represented in the right
[00:33:15.720]is what if we take a piece of DNA
[00:33:17.215]that encodes just that spike protein,
[00:33:19.770]no other part of the virus,
[00:33:21.210]we deliver it to our host cells,
[00:33:22.800]to our human cells,
[00:33:23.940]they read that chapter, that gene, that information,
[00:33:26.588]just like they would read in the other gene,
[00:33:28.590]and they express it as messenger RNA,
[00:33:30.810]produce the spike protein,
[00:33:32.280]which is then presented and results in an immune response
[00:33:35.310]and the antibodies that we hear about so often in the news,
[00:33:37.920]as well as T-cell responses.
[00:33:40.740]And so that is the approach
[00:33:43.050]that the field started to rapidly take.
[00:33:45.090]You can synthesize these very quickly
[00:33:47.370]and we knew the sequence of the spike protein
[00:33:49.380]and we knew the spike protein should be our target.
[00:33:52.260]But there is actually a couple ways
[00:33:53.640]that we could accomplish this.
[00:33:54.990]One is the way that I've been talking about
[00:33:56.760]this entire lecture,
[00:33:57.630]which is the idea that you take a piece of DNA, the gene,
[00:34:00.480]you deliver it to a cell,
[00:34:01.770]it has to get all the way to the nucleus,
[00:34:03.660]expressed messenger RNA expressed as a protein.
[00:34:06.300]And indeed this was seen in three of the vaccines
[00:34:10.800]that are being used today,
[00:34:11.910]including the AstraZeneca vaccine,
[00:34:13.740]the Johnson & Johnson vaccine,
[00:34:15.390]as well as a newer vaccine
[00:34:16.590]that was approved for use in India.
[00:34:18.600]The Johnson & Johnson and AstraZeneca vaccines
[00:34:20.640]make use of the adenovirus,
[00:34:22.290]the same virus I've been talking about
[00:34:23.790]in many of the clinical trials that have led up to today,
[00:34:26.700]to deliver the gene that encodes the S protein
[00:34:29.400]or the spike protein of the coronavirus,
[00:34:31.500]whereas the vaccine that was approved for use in India
[00:34:34.543]actually is just a circular piece of plasma DNA
[00:34:37.620]injected into the muscles.
[00:34:39.750]So that's one way to do it.
[00:34:40.980]And you can imagine,
[00:34:42.150]for everything I've talked about
[00:34:43.470]previous to vaccine applications,
[00:34:45.960]that's a good idea.
[00:34:46.800]We'd actually like the gene
[00:34:48.000]to maybe be around for a little bit in the nucleus
[00:34:50.490]to allow for the therapy to be produced
[00:34:52.260]over a long period of time.
[00:34:53.580]But for a vaccine, we don't actually need that.
[00:34:55.980]We can actually have that protein made very quickly,
[00:34:58.920]and the immune response happened very quickly thereafter.
[00:35:01.350]And so some people started to think,
[00:35:03.247]"Well, what if we kind of circumvent
[00:35:05.158]parts of the central dogma
[00:35:06.930]and we just go right in with messenger RNA?"
[00:35:09.000]Remember, DNA goes to messenger RNA, goes to protein.
[00:35:11.640]What if we just deliver messenger RNA?
[00:35:13.500]That still has the code to instruct the cell
[00:35:16.170]to make the S protein,
[00:35:17.520]but we don't have to worry about getting it to the nucleus,
[00:35:20.430]and the cell's gonna see that messenger RNA
[00:35:24.060]in any way that it would see one of its own messenger RNAs,
[00:35:26.460]all the thousands that it's processing
[00:35:29.010]every second of your life.
[00:35:30.570]And you might have said,
[00:35:31.597]"Well, why didn't we think of this before?"
[00:35:35.070]But I will say before I explain
[00:35:36.810]why we did think of this before
[00:35:38.520]is that that is the principle
[00:35:40.110]by which the Moderna and Pfizer vaccines,
[00:35:42.360]those that are used the most in the US,
[00:35:44.460]they both use the principle of delivering a messenger RNA
[00:35:47.340]that encodes the S protein to the cell.
[00:35:49.770]So going back to, why didn't we think of this before?
[00:35:51.990]Well, we had.
[00:35:53.670]We actually had been working on it for a very long time.
[00:35:56.400]The idea of delivering messenger RNA instead of a gene.
[00:35:59.790]So RNA instead of DNA.
[00:36:02.050]One shortcoming of that
[00:36:04.320]is something that I just mentioned, which is,
[00:36:05.760]in many times, you actually would like the instructions
[00:36:07.920]to stay around for a long time in the nucleus.
[00:36:10.080]In that case, you'd like to deliver a gene.
[00:36:12.000]But there's also a lot of applications
[00:36:13.590]where you don't need such as vaccine.
[00:36:15.630]However, what's something I didn't mention
[00:36:17.340]about messenger RNA earlier
[00:36:19.350]is that messenger RNA is very fragile,
[00:36:22.590]and so it is intended
[00:36:23.970]to be around for a short period of time,
[00:36:26.220]be translated to a protein, and degraded,
[00:36:28.170]so the cell can rapidly respond to changing conditions.
[00:36:31.110]And so even when we handle messenger RNA in the lab,
[00:36:34.830]it's so fragile it can degrade
[00:36:36.450]before you even have time to process it.
[00:36:38.847]And so there was a lot of technical details
[00:36:41.188]that had to be worked out and had been worked on for decades
[00:36:44.670]to try to understand how we could stabilize messenger RNA
[00:36:47.970]and enable it to be delivered.
[00:36:49.620]And so the work that I'm not gonna highlight here
[00:36:51.840]was individuals that worked
[00:36:53.280]on adding various features to messenger RNA
[00:36:56.040]so that it wouldn't degrade quite as quickly.
[00:36:58.800]But others that worked in the field,
[00:37:00.180]including Katalin Kariko and Drew Weissman,
[00:37:02.820]who came together first at the University of Pennsylvania,
[00:37:05.460]they worked for decades to pioneer mRNA technology
[00:37:08.970]to enable its use as a therapy really focusing on the idea
[00:37:12.270]that if you did try to deliver messenger RNA,
[00:37:14.220]oftentimes it would initiate an immune response.
[00:37:16.770]And so in 2005, they published this really beautiful work
[00:37:18.947]where they were able to show
[00:37:21.150]that if they modified some of the chemistry
[00:37:23.220]of the letters of messenger RNA,
[00:37:25.050]they didn't get that response.
[00:37:26.940]And so they actually had been working in messenger RNA,
[00:37:30.759]they were in a clinical trial for using messenger RNA
[00:37:34.050]for influenza in 2020
[00:37:36.270]when it became clear that we needed some technology to pivot
[00:37:40.860]and be used for the COVID-19 vaccine.
[00:37:43.320]And so, a lot of times,
[00:37:45.090]I think people think that messenger RNA
[00:37:46.800]was a new technology,
[00:37:48.060]but it was one that was being worked on famously
[00:37:50.400]by many really outstanding scientists for decades.
[00:37:53.700]Now, in addition to to settling on the idea
[00:37:56.130]of messenger RNA,
[00:37:57.000]we also had to figure out how to get it inside the cell.
[00:37:59.730]Now, just because people had worked on stabilizing it
[00:38:02.201]and making it less immunogenic
[00:38:04.050]didn't mean it still didn't need to be protected
[00:38:06.180]during its delivery.
[00:38:07.260]And so this, we went to non-viral gene delivery.
[00:38:09.570]Finally, my field got to have its day.
[00:38:12.510]Because there was some materials
[00:38:14.040]that worked very well to encapsulate
[00:38:16.292]and protect messenger RNA.
[00:38:18.300]And so if you remember back to earlier on in my talk
[00:38:21.000]when I talked about Felgner
[00:38:22.380]designing a cationic or a charged lipid,
[00:38:26.130]we went back to that work
[00:38:27.270]and actually had never left that work.
[00:38:28.710]In fact, we had been investigating lipids
[00:38:30.560]as the delivery vehicle for DNA and RNA
[00:38:33.750]back to the 1980s starting with Felgner.
[00:38:36.060]And these are basically fat bubbles
[00:38:38.786]that we know can encapsulate medicines and deliver them.
[00:38:42.630]And in fact, the FDA has approved these lipid nanoparticles
[00:38:46.314]for use for small molecules since 1995.
[00:38:49.830]This table actually represents
[00:38:51.240]many of the common medications
[00:38:53.220]that are administered using a lipid nanoparticle.
[00:38:56.250]They also, at the time of the pandemic,
[00:38:58.410]at the time of the beginning of the pandemic
[00:39:00.030]were in use for clinical trials for nucleic acids or RNA.
[00:39:03.630]And so a lot of this felt new to the general population,
[00:39:07.620]but to those of us in this field, we thought,
[00:39:09.787]"Well, we've been working on messenger RNA for a long time,
[00:39:12.600]we've been working on lipid nanoparticles for a long time,
[00:39:15.000]and it made sense then that the FDA
[00:39:18.090]working with many, many scientists
[00:39:20.580]approved two COVID-19 vaccines
[00:39:22.770]that deliver messenger RNA using lipid nanoparticles.
[00:39:26.730]And so as of two weeks ago when I pulled this data,
[00:39:29.791]there are 11 COVID-19 vaccines granted emergency use
[00:39:33.535]by the World Health Organization,
[00:39:35.520]and six of the 11 of those vaccines
[00:39:38.550]use DNA or RNA as its cargo.
[00:39:42.270]That gives me chills to say
[00:39:43.680]that this is a field I've been in my entire academic career
[00:39:46.650]and over half of the COVID-19 vaccines
[00:39:49.135]make use of technology that in the late '90s we worried
[00:39:53.130]wasn't gonna be able to reemerge back into a market.
[00:39:56.130]But now we've known that not only are they safe,
[00:39:58.740]but they are incredibly efficient and efficacious.
[00:40:02.850]And so the future and challenges of gene therapy.
[00:40:05.160]First, the future is bright.
[00:40:07.405]Efficacy has been demonstrated
[00:40:09.420]for gene replacement therapies that are now FDA approved
[00:40:12.390]and vaccines that have been injected into millions of arms.
[00:40:16.710]In just the last two years,
[00:40:18.210]there's been hundreds of applications to the FDA now
[00:40:21.180]for gene therapy products
[00:40:22.620]because of the establishment of safety delivery
[00:40:27.060]and other technological investments that we've had.
[00:40:30.390]It's estimated that the FDA will start to approve
[00:40:32.970]10 to 20 gene therapies per year by the year 2025,
[00:40:37.560]and this is after we went 30 years with only three approved.
[00:40:41.040]So this is gonna be an amazing product space.
[00:40:44.730]In addition, there'll be new advances
[00:40:46.350]in gene editing technologies like CRISPR
[00:40:48.780]that are gonna revolutionize cell and gene therapy.
[00:40:50.940]And I don't have time today to even talk about CRISPR.
[00:40:53.370]But CRISPR makes use
[00:40:54.300]of a lot of what I've been talking about today.
[00:40:56.430]And instead of delivering a gene,
[00:40:58.110]it delivers factors and mechanisms
[00:41:01.740]that can actually go in and cut part of a gene
[00:41:03.990]and edit it in real time
[00:41:05.215]rather than having to deliver the gene.
[00:41:07.650]And so the future is so bright.
[00:41:09.720]And I like to tease my students,
[00:41:12.090]it's suddenly cool again
[00:41:13.350]to be working in the area of gene therapy.
[00:41:16.050]But the challenges remain.
[00:41:17.460]And one that I talked about earlier
[00:41:18.747]and that I'll bring up again, is cost.
[00:41:21.360]All of these range from 400,000 to now being,
[00:41:25.420]unfortunately, winning the award
[00:41:27.450]of the most expensive medicine in the world.
[00:41:29.490]And that's because these medicines
[00:41:32.040]cost a lot of of money to manufacture,
[00:41:34.800]they've cost a lot of money in R&D,
[00:41:37.020]and our insurance and healthcare systems
[00:41:39.990]aren't set up well to price cures.
[00:41:43.380]And so we have to address these economic challenges.
[00:41:46.650]And one way to do that
[00:41:48.000]might be through better delivery vehicles.
[00:41:50.670]And so we've talked about viral vectors,
[00:41:52.380]we've talked about non-viral materials,
[00:41:54.420]but those two fields are still working at a rapid pace
[00:41:58.860]to try to develop better delivery vehicles
[00:42:01.410]that are more efficient.
[00:42:03.060]'Cause if they're more efficient,
[00:42:04.020]we can actually deliver less of the medicine to a person,
[00:42:07.230]and that can reduce the cost.
[00:42:08.730]And so I wanna end with a few comments
[00:42:11.550]about what my lab is working on
[00:42:13.290]in terms of new and and innovative materials
[00:42:16.770]for gene delivery.
[00:42:17.760]And this is an image of the wonderful individuals
[00:42:20.400]that I have working with me in my lab
[00:42:22.260]on engineering biomaterials for gene delivery.
[00:42:25.170]And so we do work in the non-viral gene delivery space.
[00:42:29.790]And while we have worked on lipids and polymers
[00:42:33.840]and materials, and in particular,
[00:42:36.270]we focused a lot of our efforts
[00:42:37.770]on developing techniques to deliver those genes
[00:42:41.580]using those materials to stem cells,
[00:42:44.310]we also are working on innovating new ways
[00:42:46.800]of delivering RNA and DNA.
[00:42:48.810]And one thing we're really interested in
[00:42:50.340]is using extracellular vesicles.
[00:42:52.260]And so extracellular vesicles are these vesicles
[00:42:54.780]that nearly all cells that we can find,
[00:42:57.600]including mammalian cells and bacteria cells, produce.
[00:43:00.384]So these little vesicles bud out of the cell,
[00:43:03.990]and these vesicles contain materials
[00:43:06.600]both on their inside and on their surface
[00:43:08.940]that the cells are actually trying to use these vesicles
[00:43:11.670]as a method or a mechanism
[00:43:13.650]to communicate with cells in their environment.
[00:43:16.500]And so we thought
[00:43:17.940]if cells are already using this as a delivery vehicle,
[00:43:20.550]can we use it as a delivery vehicle for cargo,
[00:43:23.940]DNA and RNA, that we want to deliver?
[00:43:26.607]And so there's different ways
[00:43:28.590]that we might accomplish this.
[00:43:29.670]In one way, we actually can deliver
[00:43:31.590]what we're interested in delivering in the vesicle
[00:43:34.080]to the host cell or the producer's cell,
[00:43:36.270]and then that car goes loaded into the extracellular vesicle
[00:43:39.330]that buds off and you can collect it,
[00:43:41.130]and the cell has done your loading for you.
[00:43:43.410]Or you might collect these formed vesicles
[00:43:45.666]that have bud off cells,
[00:43:47.400]and then manipulate them
[00:43:48.990]through physical or chemical techniques
[00:43:51.030]to load your DNA or RNA into these vesicles.
[00:43:53.934]And we're actually exploring
[00:43:55.590]both of these approaches in my lab.
[00:43:57.540]For the former,
[00:43:58.373]we are interested in using human mesenchymal stem cells
[00:44:01.440]as the producer of the extracellular vesicle.
[00:44:03.960]So these cells are found in bone marrow and fat tissue,
[00:44:07.620]and they were famously determined
[00:44:10.020]to have differentiation potential,
[00:44:11.640]which means that if you add the right factors
[00:44:14.880]to these cells,
[00:44:15.713]they can become bone cells or fat cells or cartilage cells.
[00:44:18.480]But they also, in just their state pre-differentiation,
[00:44:22.440]they have immunomodulatory capacity,
[00:44:24.390]which basically has been shown
[00:44:26.490]that if you administer these cells
[00:44:28.200]to various parts of the body,
[00:44:29.700]and this has been done in many clinical trials,
[00:44:32.310]they can aid healing or aid in a disease response.
[00:44:35.760]And it's actually thought
[00:44:37.500]that it's not the cells necessarily themselves
[00:44:39.960]that's doing this, but it's their extracellular vesicles
[00:44:42.690]that are budding off of those cells
[00:44:44.370]that are mediating these positive effects
[00:44:47.190]seen in clinical trials.
[00:44:48.330]And oftentimes that's because of the cargo
[00:44:51.270]that these cells are loading themselves
[00:44:53.370]and also some of the features that they're placing
[00:44:55.440]on the outside of these extracellular vesicles.
[00:44:57.600]So in our lab, we've actually developed a technique
[00:45:00.270]where we use DNA,
[00:45:01.920]a plasma that has many genes on it,
[00:45:04.020]and we transback that, that's the word we use,
[00:45:06.150]into these stem cells.
[00:45:08.040]And what these genes,
[00:45:09.900]these instructions that we're delivering do,
[00:45:11.940]is they tell the stem cell
[00:45:13.200]that every time it makes an extracellular vesicle
[00:45:15.110]or we call them exosomes from mammalian cells,
[00:45:17.850]that they load something that we have encoded on that plasma
[00:45:21.360]that we want an RNA that we want loaded.
[00:45:23.700]And so the idea is that,
[00:45:25.800]we are using sort of the magic of the exosomes,
[00:45:28.560]of these mesenchymal stem cells,
[00:45:30.090]but then endowing them with the cargo,
[00:45:32.190]the RNA that we want to deliver.
[00:45:34.290]We also are decorating these exosomes
[00:45:36.330]with targeting ligands, sort of like the CAR T-cells,
[00:45:39.120]which allow them to target to a disease state.
[00:45:41.610]And we're investigating these exosomes right now
[00:45:44.490]in therapy for cardiovascular disease.
[00:45:47.820]Finally, we're also interested
[00:45:49.260]in loading preformed extracellular vesicles.
[00:45:51.510]And in this, we're really interested
[00:45:52.920]in using extracellular vesicles from bacteria.
[00:45:56.100]And this work is in collaboration
[00:45:57.660]with Dr. Amanda Ramer-Tait
[00:45:58.950]in the Food Science and Technology Department.
[00:46:00.930]And the idea here is that we actually wanna use these
[00:46:03.420]for oral DNA delivery.
[00:46:05.250]So everything I've talked about so far today,
[00:46:07.020]we either take cells out of a patient,
[00:46:08.850]deliver genes and give those cells back,
[00:46:10.800]we inject through IV or intramuscularly,
[00:46:15.180]but oral gene delivery would allow us
[00:46:17.580]to treat various diseases within the gut,
[00:46:20.130]it would also allow us to potentially mediate vaccination,
[00:46:23.850]and there's a lot of advantages to oral gene delivery
[00:46:26.490]or oral delivery of any drug,
[00:46:27.990]and that you have high patient compliance.
[00:46:29.760]Many people would rather take an oral medication
[00:46:32.070]than have a shot.
[00:46:33.210]We don't have to have medical personnel for administration,
[00:46:35.820]we have a large surface area
[00:46:37.620]where we can deliver the medicine,
[00:46:39.510]and we can either have a local or a systemic response.
[00:46:41.940]However, there are not a lot of oral drugs on the market
[00:46:45.510]because of the barriers within our GI tract,
[00:46:47.595]including the low pH and enzymes of the stomach,
[00:46:50.880]as well as a mucus layer
[00:46:52.530]and other enzymes within the intestine.
[00:46:55.680]But we think that we can use these budding vesicles,
[00:46:58.813]which are called outer membrane vesicles,
[00:47:00.657]and they come from bacteria,
[00:47:02.190]we think we can use them from commensal gut bacteria.
[00:47:05.670]That is, that bacteria that are present in the human gut
[00:47:09.210]that are not causing disease.
[00:47:11.100]And we know that these bacteria that reside in your gut
[00:47:15.000]make these vesicles.
[00:47:16.140]These vesicles contain various biomolecules,
[00:47:18.840]they are used for cell to cell communication,
[00:47:21.171]they also can cross the mucosal barrier
[00:47:23.490]within your intestine and be internalized
[00:47:25.560]by the human intestinal epithelial cells.
[00:47:28.230]And so what we're doing right now
[00:47:29.790]is we're developing the techniques
[00:47:32.190]to grow up these bacteria in the lab,
[00:47:35.340]collect the extracellular vesicles that they are secreting,
[00:47:38.370]load our DNA into them, and then deliver them orally
[00:47:42.180]either for therapy or vaccination applications.
[00:47:44.760]And our early data suggests that we,
[00:47:46.416]in mouse models, that we have been able to show
[00:47:49.170]that we can transfer a gene using these vesicles.
[00:47:52.680]And so I hope that my lecture today has shown you
[00:47:57.090]that the history has not...
[00:48:00.581]Has been rocky at times
[00:48:03.320]within the field of DNA and RNA delivery,
[00:48:06.690]but we have worked hard as a community
[00:48:09.090]to understand how to safely deliver both genes and RNA
[00:48:14.460]and to do these in a way that is really proving
[00:48:17.460]or will prove to impact the therapeutic space.
[00:48:20.220]And in fact, some have said that this will be
[00:48:22.620]the fastest growing area of therapeutics
[00:48:25.020]that's really estimated to go
[00:48:26.670]from a $6 billion market last year,
[00:48:29.310]to almost 43 billion market by 2030,
[00:48:32.985]which is only eight years from now.
[00:48:35.070]And so it's an exciting time to be in this field,
[00:48:38.220]it's exciting time to be a patient
[00:48:40.860]that might have their life dramatically changed
[00:48:43.920]by these therapies.
[00:48:45.240]With that, I'd just like to thank my lab
[00:48:47.010]for the small amount of research that I spoke of at the end,
[00:48:49.680]my collaborators, my funding sources,
[00:48:51.630]and, again, to Chancellor Ronnie Green
[00:48:54.300]and Vice Chancellor Bob Wilhelm for the invitation to speak.
[00:48:56.940]Thank you so much.
[00:49:04.530]Wow, Angie, that was absolutely fantastic.
[00:49:07.410]Thank you so much for the historical tour
[00:49:10.020]through the history of gene delivery therapy
[00:49:13.350]as well as what you're doing up to now in your current lab.
[00:49:16.410]So thank you so much for that.
[00:49:17.670]Thank you.
[00:49:18.503]It was fun to put it together and describe
[00:49:20.400]what's a phenomenal history and a really exciting future.
[00:49:24.558]Yeah.
[00:49:25.391]And I love that you inserted a little history from Nebraska.
[00:49:27.143]That's right.
[00:49:28.110]That was really exciting.
[00:49:29.940]An important part.
[00:49:31.050]Yeah, so we have lots of questions,
[00:49:32.743]and I'm gonna open up with the first one,
[00:49:35.190]which is, "How will the new mRNA vaccines
[00:49:39.045]change future therapeutics?"
[00:49:41.370]Yeah, that's a great question
[00:49:42.750]and something that we're really excited about
[00:49:44.625]within our field.
[00:49:45.870]And so I think that given that we've now injected
[00:49:50.057]messenger RNA vaccines, within this fat bubble,
[00:49:53.910]within this lipid nanoparticle
[00:49:55.110]into millions or billions of people,
[00:49:57.480]we have demonstrated safety and efficacy of this system.
[00:50:02.040]And the idea is that,
[00:50:03.510]since messenger RNA is simply a recipe,
[00:50:05.610]it's simply a sequence,
[00:50:06.630]we can change that recipe for a different protein
[00:50:10.020]that might have a different therapeutic target
[00:50:12.120]and we can demonstrate like with FDA approval
[00:50:15.900]and other types of routes
[00:50:17.670]or other types of parts of an application you have to do
[00:50:20.970]to get a drug approved,
[00:50:21.960]that we have safety and efficacy proven with the system,
[00:50:23.985]with the lipid nanoparticle and with the messenger RNA.
[00:50:27.240]So I think that sets us so far ahead
[00:50:29.370]because we've done that back...
[00:50:31.260]Not really background work,
[00:50:32.460]but it's was done under emergency use authorization,
[00:50:36.180]and now approved authorization for those vaccines.
[00:50:39.090]We had been in the field working on messenger RNA
[00:50:41.910]for cancer therapeutics, for other vaccines,
[00:50:44.700]including influenza.
[00:50:46.200]And a lot of lessons have been learned
[00:50:48.593]about how to design that system.
[00:50:51.030]And so I'm really excited.
[00:50:52.110]I think that it's shown
[00:50:54.210]that that type of delivery system can work.
[00:50:57.030]I will admit that the messenger RNA vaccines,
[00:51:00.300]you don't have to have great expression
[00:51:01.890]to have a good immune response and a good vaccine response.
[00:51:04.710]When we start thinking about things like cancer
[00:51:06.960]or other therapies,
[00:51:07.830]we do have to increase our delivery efficiency,
[00:51:10.650]which is why I sort of ended the talk with other vesicles
[00:51:13.380]and other types of delivery carriers
[00:51:15.030]because that still is a hurdle that we'll have to overcome
[00:51:18.000]as we move forth into other therapies
[00:51:20.760]or other therapeutic targets.
[00:51:22.200]But I think that the precedence that's been set up
[00:51:25.080]is gonna allow for a really bright future
[00:51:26.790]with those therapies.
[00:51:28.920]Just to follow up,
[00:51:30.420]will this replace or be additional to the current,
[00:51:35.280]more traditional medicines? Sure, yeah.
[00:51:36.900]I think I've seen it and we've proposed it in both ways
[00:51:40.140]and so it may completely replace
[00:51:41.670]some of our standard therapeutics
[00:51:43.740]because they're just so much more precise.
[00:51:46.200]They're actually getting after, potentially,
[00:51:49.800]the causative agent of the disease.
[00:51:51.960]But I've also seen some really beautiful studies
[00:51:53.820]where we use it in combination with,
[00:51:55.532]for example, for cancer with existing chemotherapies
[00:51:59.215]in sort of as the combinatorial type of treatment.
[00:52:02.130]But I think with gene therapy,
[00:52:05.100]we did have this decade long pause from the late 1990s
[00:52:09.750]to really, you know, 2010, 2015 where we learned so much.
[00:52:15.000]And now with the vaccine work of the last three years,
[00:52:18.300]we've learned an incredible amount of information
[00:52:20.460]that's really gonna set us up
[00:52:21.600]for some really, really fantastic future therapies.
[00:52:25.290]Oh, that's great.
[00:52:27.180]So something I've always wondered about,
[00:52:29.880]but with very superficial knowledge, don't have the answer,
[00:52:32.610]and so I can't wait to hear it from you is,
[00:52:33.710]are all the gene therapies meant
[00:52:35.970]to be permanently changing the genome of the patients?
[00:52:39.750]Absolutely not.
[00:52:40.650]I mean, when we're going after a genetic disorder,
[00:52:44.580]we are gonna want the gene to be remain in the nucleus
[00:52:47.820]for a period of time.
[00:52:48.690]That's important for the therapy,
[00:52:50.608]which, in some cases, may be for the rest of the life
[00:52:53.010]of the patient.
[00:52:53.970]But for vaccines for instance,
[00:52:55.650]we don't need it around very long at all.
[00:52:57.807]We just need it to be expressed
[00:53:00.390]and the immune response to be generated,
[00:53:02.220]and then the gene doesn't have to be there any longer.
[00:53:04.530]And so we have to look at different ways of delivering genes
[00:53:07.680]based on how long we want that gene to be around the nucleus
[00:53:10.710]or how long we need the messenger RNA to be there
[00:53:13.590]as a recipe for the protein.
[00:53:15.120]But, no, certainly not all of them
[00:53:16.920]have to be integrated is what we would call,
[00:53:19.230]but some we do have to have that happen, so...
[00:53:22.620]So how do we make that decision then?
[00:53:24.600]Right.
[00:53:25.433]Well, it has a lot to do with where in the lifespan
[00:53:28.907]of your patient you're applying the therapy.
[00:53:30.789]So the new very famously expensive medication
[00:53:34.495]or gene therapy is designed for infants,
[00:53:37.740]and so that is given in an infant stage
[00:53:40.350]and we want that to be curative,
[00:53:42.000]so we need that gene to remain in the patient
[00:53:44.160]for the rest of their life.
[00:53:46.680]Wow.
[00:53:47.749]So what technologies beyond what you've described,
[00:53:54.450]do you think we need for gene therapy to be successful?
[00:53:58.320]Well, I...
[00:53:59.340]So just to reemphasize,
[00:54:02.100]I think that we have to keep working
[00:54:04.530]on better delivery systems.
[00:54:06.420]And so as I described for a lot of the vaccine work,
[00:54:10.860]we need it to...
[00:54:11.693]We inject it intramuscularly,
[00:54:13.440]the cells take up that messenger RNA or the gene,
[00:54:16.200]they express enough of that protein
[00:54:17.850]to have a robust immune response.
[00:54:19.740]But if we're trying to treat some genetic disorder
[00:54:22.050]that affects every muscle fiber in your body
[00:54:24.300]or every lung cell in your lung,
[00:54:26.209]we need that to be much more efficient
[00:54:28.734]than just a few cells in your arm.
[00:54:30.900]And so we have to have better delivery vehicles
[00:54:33.300]that allow for that.
[00:54:34.260]Also to allow for targeting.
[00:54:35.700]We may want to systemically inject it through an IV
[00:54:38.790]and it may circulate through your whole body,
[00:54:40.770]but we only want that gene to be delivered to the heart,
[00:54:43.590]say, for instance.
[00:54:44.580]And so targeting and delivery, I think, is really important.
[00:54:49.080]And then the other technologies is just...
[00:54:52.230]With CRISPR editing,
[00:54:53.220]which I talked about briefly at the end of my talk,
[00:54:56.220]that's an opportunity not to deliver the gene,
[00:54:58.710]but to sort of deliver some scissors and some paste
[00:55:01.410]to take out the gene and put in the gene
[00:55:03.808]that you're trying to correct the mutation in.
[00:55:08.610]That we need to work on, targeting and safety as well.
[00:55:12.445]So where is CRISPR gonna be like in...
[00:55:15.863]And I know where it is now,
[00:55:17.310]you just helped me understand that,
[00:55:18.719]but where is it going to be?
[00:55:19.905]Well, CRISPR is a very exciting part of gene therapy.
[00:55:23.678]I didn't have a lot of time to talk about that today.
[00:55:26.457]It, in some way, is similar in that
[00:55:28.620]rather than delivering a gene,
[00:55:29.880]you deliver gene or genetic components
[00:55:32.100]that give the cells instructions to edit their genes
[00:55:34.770]in a way that's safe.
[00:55:36.920]So that...
[00:55:38.697]And some of the concerns we have
[00:55:40.290]talking about delivering a gene to a nucleus,
[00:55:42.630]and does it need to stay around long?
[00:55:44.280]This would answer that question.
[00:55:45.360]You would actually edit the patient's own gene
[00:55:47.460]that's already the book chapter on the chromosome.
[00:55:50.100]And so I think it will have a place
[00:55:51.780]that's certainly in clinical trials,
[00:55:53.280]and there's been some interesting
[00:55:54.960]and great results with safety
[00:55:56.300]on some various cancer treatments
[00:55:59.370]and other types of treatments as well.
[00:56:01.677]And so I think that the issues with CRISPR
[00:56:05.310]are mainly that while the scissors
[00:56:07.200]and the cutting and the pasting are specific,
[00:56:09.622]they can have off target effects.
[00:56:11.217]And so we have to make sure
[00:56:12.390]that we understand what that means
[00:56:14.640]and how do we mitigate those off target effects
[00:56:16.770]so that we don't have any cutting of the genome
[00:56:18.540]anywhere where we don't want cutting.
[00:56:20.700]But I think it's...
[00:56:21.533]You're gonna see CRISPR in everything.
[00:56:23.294]From animals for food production, to plants, to therapies.
[00:56:27.878]It's gonna really change the way
[00:56:30.600]that we go about delivering genes or modifying genes
[00:56:33.560]in a variety of organisms.
[00:56:36.240]So if I understood that, and maybe not,
[00:56:38.400]so if there's like changes in gene
[00:56:41.370]through experience methylation,
[00:56:42.808]will it be able to think about that?
[00:56:45.188]Yeah, so in terms of methylation or acetylation,
[00:56:47.870]that's more epigenetic modifications.
[00:56:50.790]You can use CRISPR to also modify the epigenome,
[00:56:54.330]which is not what I talked about today.
[00:56:56.100]No, no, no. Good job Dr. Bevins.
[00:56:58.890]So epigenetics is the idea that the sequence of bases
[00:57:03.330]is not the only thing that's important to control a gene.
[00:57:06.060]There's also little knobs that we can kind of place
[00:57:08.580]either on the gene itself or on the proteins
[00:57:11.680]that genes are wrapped around within a chromosome.
[00:57:15.330]And that helps to keep genes on or off.
[00:57:18.076]So the book chapters red or not red.
[00:57:20.580]And so there's actually ways that you can modify CRISPR,
[00:57:23.160]and rather than cutting,
[00:57:24.750]you just use it to sort of dock near a gene
[00:57:26.880]where you need it to be,
[00:57:27.750]and then it can put on various acetyl groups
[00:57:29.880]or methyl groups as well, so...
[00:57:32.250]Wow, nice. Yeah, Yeah.
[00:57:33.420]It's very exciting.
[00:57:34.388]It is.
[00:57:35.221]And we actually work on that in my lab,
[00:57:36.570]but I didn't talk about some of the CRISPR epigenome editing
[00:57:39.172]that we're doing.
[00:57:40.170]So, yeah, it's very exciting.
[00:57:41.850]Well, that's a perfect segue
[00:57:43.110]to two questions I want to dive in
[00:57:45.450]a little deeper about you,
[00:57:47.459]'cause you...
[00:57:48.292]We only had like just the tail end of about you.
[00:57:50.279]So I guess the first one is a sort of,
[00:57:53.089]how did you get interested in this field?
[00:57:55.380]What pulled you this way?
[00:57:56.670]Yeah.
[00:57:57.540]Well, I think, for me,
[00:57:59.299]as an undergraduate engineering student,
[00:58:02.670]I was first just interested in science.
[00:58:05.880]And so I found myself as an undergraduate researcher
[00:58:09.540]in a lab that worked on gene delivery.
[00:58:11.790]We've sort of worked on gene delivery as a tool
[00:58:13.890]to study various other things,
[00:58:16.260]and I kind of became fascinated.
[00:58:17.880]I was an engineer
[00:58:18.810]that had taken no biology courses at that time,
[00:58:21.327]and I was fascinated by the idea of DNA and it's regulation,
[00:58:25.680]and I loved it.
[00:58:28.203]Then, as you look back over your career,
[00:58:30.690]it looks like it was very intentional,
[00:58:32.250]but at the time, there was a lot of wandering.
[00:58:34.380]And I ended up in a master's lab
[00:58:36.720]that worked on DNA delivery,
[00:58:40.290]really small pieces of nucleic acids,
[00:58:42.720]and then when I found myself looking for a lab for my PhD,
[00:58:45.990]I was, of course, attracted to a lab
[00:58:47.730]that happened to work on gene delivery again.
[00:58:49.980]And so I've been fascinated with DNA for as long as,
[00:58:53.083]at least at the beginning of my career and since now.
[00:58:55.240]And I have art of DNA in my office and at my house.
[00:58:58.230]I just love it.
[00:58:59.340]I love everything.
[00:59:00.173]I think it's probably the engineer in me.
[00:59:01.800]It's a code that we can decode
[00:59:04.020]and that we can use potentially as a therapy.
[00:59:08.040]And then the other question I had to dive into
[00:59:11.550]to more about what you're working on,
[00:59:13.140]what you're thinking about is,
[00:59:15.420]so if you were to come back
[00:59:16.410]and see us in 10 years and do another lecture,
[00:59:18.780]where do you think your lab's gonna be?
[00:59:19.920]Where do you think your work's going going to be?
[00:59:21.540]Yeah.
[00:59:22.373]I think it'll be working a lot in the extracellular vesicles
[00:59:25.434]that I talked about at the end of my presentation,
[00:59:27.686]which is sort of nature's way of delivering nucleic acids
[00:59:30.693]in a similar way that viruses
[00:59:32.819]is one way that nature delivers genes.
[00:59:35.310]We also know that nature uses these extracellular vesicles
[00:59:38.190]to deliver nucleic acids,
[00:59:39.467]which are the bases of DNA and RNA.
[00:59:43.860]And so we have some exciting results in the lab
[00:59:46.497]that I shared a little bit in the lecture,
[00:59:48.450]but I think that those really have a huge potential
[00:59:51.459]to be safely harnessed as a delivery vehicle.
[00:59:54.750]So I think that's where you'd find me in 10 years,
[00:59:56.640]is I'd be sharing really exciting results
[00:59:59.220]about our oral delivery system
[01:00:00.979]or using stem cell derived exosomes
[01:00:04.497]for some sort of treatment.
[01:00:06.643]Okay.
[01:00:07.476]Nice, thank you.
[01:00:08.340]So we got a question from the audience here,
[01:00:11.700]and I'll read it and do my best job.
[01:00:14.437]"For the COVID vaccine,
[01:00:17.498]how is the viral DNA inserted into the plasmids
[01:00:21.900]that are in the first step of the vaccine production
[01:00:24.837]and where did this DNA come from?"
[01:00:27.600]Okay, that's a great question.
[01:00:29.070]So there are a couple of COVID-19 vaccines
[01:00:31.710]that do use a plasmid system
[01:00:33.547]to insert into the viral vector.
[01:00:37.440]And so that viral vectors are produced
[01:00:40.655]by taking various plasmids or pieces of DNA
[01:00:45.240]that have genes encoded on them,
[01:00:46.620]including the spike protein for the vaccine,
[01:00:48.720]but you'll also use plasmids that have genes
[01:00:50.580]that encode for proteins that package a virus.
[01:00:53.040]And you take those plasmids
[01:00:54.570]and you put them into a producer cell line.
[01:00:57.120]And that producer cell line then
[01:00:58.950]produces the virus for you.
[01:01:00.480]You collect it, purify it,
[01:01:02.670]and then you can use it as a vaccine.
[01:01:05.280]And I think that's what that question was getting at.
[01:01:07.290]In terms of the messenger RNA vaccines,
[01:01:11.700]we can actually synthesize that messenger RNA.
[01:01:14.550]There's various ways that you can take a piece of DNA.
[01:01:17.214]You can use in vitro transcription
[01:01:19.170]to actually make messenger RNA,
[01:01:20.233]so you are not even using an organism
[01:01:22.410]at that point to make it.
[01:01:23.790]And so there are steps you have to do
[01:01:27.300]to use some producer cells
[01:01:29.370]and various features to produce those vaccines,
[01:01:32.400]but the sequence,
[01:01:33.607]famously, we sequenced the SARS-CoV-2 very quickly
[01:01:37.851]because of our huge advancements in sequencing technology.
[01:01:41.274]And so we knew early on
[01:01:43.530]what the sequence of that spike protein was.
[01:01:45.960]We also knew from the MERS and the SARS outbreaks
[01:01:48.930]that the spike protein
[01:01:49.770]was something that we should focus on.
[01:01:51.300]And so we're able to get the sequence,
[01:01:54.334]and then you can clone these genes,
[01:01:56.520]which is a scary word, but it's really not.
[01:01:58.410]It just means move them around in various ways
[01:02:02.410]in terms of like shuttling them in and out of plasmids.
[01:02:05.490]Something I couldn't talk about today
[01:02:06.810]is gene therapy wouldn't be possible
[01:02:08.490]without all the recombinant DNA technology,
[01:02:10.740]which is technology that came forth
[01:02:13.080]in the '60s, '70s, and '80s,
[01:02:14.490]where we learned how to take genes,
[01:02:16.320]cut them out of an organism,
[01:02:17.760]and then place them into plasmids
[01:02:19.680]and use that to shuttle these genes around.
[01:02:22.530]So it was a great question.
[01:02:23.941]I hope that I answered it well.
[01:02:25.800]And if I didn't, whoever asked it,
[01:02:27.210]email me and we could talk more
[01:02:28.830]about how the viral vectors or the viral viruses
[01:02:32.160]were made as well as the non-viral.
[01:02:33.780]Yeah, thank you for that.
[01:02:34.770]Yeah, lots of people I know are interested in that question
[01:02:37.440]and that process.
[01:02:38.886]So another question we have is that,
[01:02:40.897]"Can you describe the techniques, methods
[01:02:43.260]of running an experiment?"
[01:02:44.640]And I'll let you interpret that
[01:02:45.680]at the level that you'd like.
[01:02:47.490]Running an experiment.
[01:02:49.140]Yeah. Yeah.
[01:02:50.070]That's a great question.
[01:02:51.330]Yeah. You and I could probably talk about this
[01:02:53.680]for a very long time of how to run an experiment well.
[01:02:57.000]A way to not run an experiment is to walk into the lab
[01:02:59.640]and decide to run an experiment.
[01:03:02.040]Experiments begin with a lot of planning
[01:03:04.212]and a lot of thinking.
[01:03:05.970]And so if we're running an experiment in my lab,
[01:03:09.060]we are usually,
[01:03:10.050]we start out and we have a protocol
[01:03:12.750]written for every experiment.
[01:03:13.830]And the very first thing we write is our hypothesis.
[01:03:16.110]So what's the objective our experiment?
[01:03:19.110]What are we hypothesizing?
[01:03:20.700]And that's really an important part
[01:03:22.230]of designing an experiment because you need to think about
[01:03:25.590]what are your variables then.
[01:03:27.480]And so you need to have variables
[01:03:29.760]in terms of your experimental variables.
[01:03:31.440]So maybe for us, maybe we're testing
[01:03:33.480]a couple different types of vesicles
[01:03:36.090]for their ability to deliver something.
[01:03:38.340]We have to have a control where we know something
[01:03:40.890]that might really be able to deliver it
[01:03:42.540]so that we know when it works, what it looks like.
[01:03:44.730]And then for us, we take those various features,
[01:03:48.690]we often will start with delivering those to cells
[01:03:51.230]in the lab.
[01:03:52.350]And for gene delivery work,
[01:03:54.780]we use a lot of genes from jellyfish.
[01:03:58.440]So we use the gene that glows green.
[01:04:00.810]We use genes from fireflies.
[01:04:02.430]So the gene that makes something glow like a firefly.
[01:04:05.100]And it is a gene,
[01:04:06.780]so it's a book chapter that encodes for a protein,
[01:04:09.810]and then we can see that protein.
[01:04:11.100]We can either see it under a microscope
[01:04:12.510]or we can see it light up like a firefly.
[01:04:14.610]So that's not a therapeutic gene.
[01:04:16.410]We'd never use that in a human,
[01:04:18.700]unless people really wanna glow, you know?
[01:04:23.040]But we use it because it's an early,
[01:04:24.960]we can indicate then in our cells,
[01:04:26.880]did we deliver the gene?
[01:04:27.960]Did it get all the way to the nucleus?
[01:04:29.490]Did it get transcribed into messenger RNA?
[01:04:31.440]Did it get translated into a protein?
[01:04:32.970]If that works, and it looks promising,
[01:04:34.740]then we typically, in my lab, work in mouse models.
[01:04:37.140]And so then you'd move into a mouse model
[01:04:39.060]and look for an effect there
[01:04:41.400]for our oral gene delivery system.
[01:04:42.960]That means orally delivering those to mice
[01:04:45.090]and looking for various features within both their intestine
[01:04:48.509]as well as perhaps their fecal matter.
[01:04:50.880]So, yeah, that's sort of...
[01:04:52.710]It's very glamorous.
[01:04:54.318](both laughing)
[01:04:57.030]Yeah, nice. Yeah.
[01:04:58.140]Didn't think we'd sneak in that word in the-
[01:05:00.840]Yeah, yeah.
[01:05:02.850]I have some sort of lisp.
[01:05:03.780]I'm trying to sneak in certain words, right?
[01:05:06.690]No, thanks for that.
[01:05:07.523]It's very...
[01:05:10.440]Taking the veil off
[01:05:11.520]of some of the experiments you're working on.
[01:05:13.200]I appreciate that.
[01:05:14.550]Another question we have is the,
[01:05:16.237]"Are the chemical chemical structures of the targeting RNA
[01:05:19.260]identical to the naturally or occurring bases,
[01:05:22.350]or are they modified in some way?"
[01:05:23.950]Yeah, that's a great question.
[01:05:25.470]So I spoke about it briefly within the lecture,
[01:05:28.380]but the messenger RNA that's used in the COVID-19 vaccines
[01:05:31.380]is chemically modified.
[01:05:33.090]And it's chemically modified to improve its stability
[01:05:37.140]'cause as I said, RNA is very degradable.
[01:05:39.150]If you look at it in the lab, it will degrade.
[01:05:41.010]And so, in the field, we were able to identify
[01:05:44.910]some various features of RNA
[01:05:46.980]that you need to add to help its stability,
[01:05:49.710]and then some of the bases
[01:05:51.180]do have some chemical modifications
[01:05:52.950]to reduce their immune response.
[01:05:55.980]And how are those decisions made on the modifications?
[01:05:58.830]Well, that was some of the early work that...
[01:06:00.563]Or the work by Weissman et al. in 2005
[01:06:04.314]that showed that you had to make those modifications
[01:06:07.230]so that you could reduce the immune response
[01:06:09.720]of delivering messenger RNA.
[01:06:13.294]A question I've been thinking about just came in.
[01:06:16.856]I love this question.
[01:06:17.880]Loved all the questions.
[01:06:18.937]"What are some ethical considerations, if any,
[01:06:21.660]that come up in your research?
[01:06:23.460]What guide your responses to those?"
[01:06:25.170]Yeah, that's a great question.
[01:06:27.510]So the idea of...
[01:06:28.966]I also work in stem cells,
[01:06:30.630]the idea of stem cells and gene therapy.
[01:06:32.430]There are obviously ethical considerations,
[01:06:37.020]and these range from,
[01:06:39.090]should we deliver genes that modify individuals
[01:06:43.080]to make them maybe better than they are?
[01:06:45.330]Like, we're talking a lot about delivering genes
[01:06:47.280]to cure diseases or to treat diseases
[01:06:48.966]or to protect against diseases, right?
[01:06:52.260]But what about delivering genes
[01:06:53.790]to give someone some superhuman strength or intelligence?
[01:06:57.495]What about the idea
[01:06:58.903]that these technologies that we're developing
[01:07:02.250]are cost prohibitive
[01:07:03.270]to the majority of the global population?
[01:07:05.490]And so I do think that ethics comes in
[01:07:08.299]to a lot of what we do.
[01:07:11.700]Dare I would say that we take that very seriously
[01:07:14.976]in the gene therapy community
[01:07:17.140]and have agreements on what we will and will not work on
[01:07:20.490]in terms of modification to certain types of cells
[01:07:23.760]or using certain types of modifications.
[01:07:26.190]And I think too that, for me,
[01:07:28.117]the ethics of cost that will hopefully,
[01:07:32.400]as we come up with better delivery systems
[01:07:34.230]and better manufacturing practices,
[01:07:36.060]we can reduce that cost
[01:07:37.290]like we do with a lot of technologies.
[01:07:39.930]But, yeah, for me,
[01:07:40.860]I guess there's some black and white boundaries of ethics,
[01:07:48.030]but then there's a lot of what we do,
[01:07:49.440]which is we're trying to help individuals.
[01:07:51.690]We're trying to help people have longer, healthier lives.
[01:07:54.750]Or in a case of pediatric patients,
[01:07:56.436]we're trying to give them a chance at life.
[01:07:58.590]And so I think that this technology,
[01:08:03.660]we know how to do it safely,
[01:08:05.730]the FDA has been a partner in this community for a long time
[01:08:08.970]with how to make sure
[01:08:10.320]that we are having the right checkpoints
[01:08:12.390]and clinical trials.
[01:08:14.220]And so I guess what guides me is that,
[01:08:16.440]in the end, I really want to help people
[01:08:18.240]and create new therapies that are giving people,
[01:08:21.363]in some cases, curing them.
[01:08:25.140]Are there groups or societies
[01:08:28.830]that sort of think about these issues and advise
[01:08:31.380]or work with scientists? Yeah, absolutely.
[01:08:33.040]Yeah, yeah.
[01:08:33.873]And so, I mean, within CRISPR just itself,
[01:08:36.180]there's certain advisory groups that have agreed for,
[01:08:38.360]we will not CRISPR edit embryos,
[01:08:40.530]we will not do certain things.
[01:08:42.150]And so, yeah, there's definitely organizations
[01:08:45.600]that, together with the FDA in the US, the NIH,
[01:08:49.950]that have agreements on how do we handle recombinant DNA,
[01:08:52.838]how do we handle everything?
[01:08:54.518]Even here at the university,
[01:08:56.220]we have incredible committees and protocols and procedures
[01:09:00.823]to ensure the safety not only of our lab personnel,
[01:09:04.020]but also the safety of the public
[01:09:05.773]so that nothing can get out from the lab
[01:09:08.190]or anything like that.
[01:09:09.023]So I think...
[01:09:11.070]We take that all very seriously.
[01:09:13.080]Yeah, no, thank you for that.
[01:09:14.647]"So it seems there was a funding lull
[01:09:17.280]between the Human Genome Project and COVID-19.
[01:09:20.700]Was the pandemic a massive boon to research?"
[01:09:24.690]That's a great question,
[01:09:26.010]and I'm not sure I'm completely prepared to answer that.
[01:09:28.961]That's a tricky one.
[01:09:30.650]I guess it would depend on who's thinking
[01:09:34.410]there was a funding lack or boom.
[01:09:36.090]I mean, the NIH was certainly investing in many things
[01:09:39.804]from Human Genome,
[01:09:41.640]which was unveiled in early 2000s,
[01:09:44.220]2003 until 2020 for sure.
[01:09:47.550]There was definitely investments in a variety of therapies.
[01:09:51.133]Maybe there was less investment
[01:09:53.730]by some venture capitalists in groups
[01:09:56.160]on gene therapy for a period of time,
[01:09:59.640]but, yeah, I think that, you know,
[01:10:02.970]I'll say, the COVID-19 vaccine shows
[01:10:04.920]that if you've put an unlimited amount of money
[01:10:06.810]towards a problem, you can cure and solve it pretty quickly.
[01:10:09.930]Cure might be a strong word,
[01:10:10.860]but you can definitely develop a vaccine
[01:10:13.380]that can change the course of a pandemic very quickly.
[01:10:15.802]I think even those of us in the community
[01:10:19.320]that have worked on lipid nanoparticles,
[01:10:21.300]that work on nucleic acid delivery,
[01:10:24.090]wouldn't have believed
[01:10:25.530]that from sequencing the SARS-CoV-2 virus in January, 2020,
[01:10:30.510]to vaccines in arms by the end of 2020,
[01:10:34.290]that we could do that.
[01:10:35.880]But it turns out,
[01:10:36.750]if you invest a lot of money
[01:10:38.040]and you put a lot of time and resources,
[01:10:40.260]both at the FDA but also almost any lab I knew
[01:10:42.840]that was working in this space,
[01:10:43.950]they were working around the clock,
[01:10:46.740]that we can really, really come out
[01:10:49.350]with some cool technology that is very effective.
[01:10:52.140]So, imagine what else we can do with continued investment
[01:10:54.791]in our research.
[01:10:56.256](Dr. Pannier laughing)
[01:10:57.300]I do think history's gonna remember this
[01:10:59.310]as a stunning example of how you don't know exactly
[01:11:03.030]why you're going to need the fundamental science-
[01:11:05.880]Absolutely. But then you need it.
[01:11:08.070]Yeah, famously, when the modified messenger RNA
[01:11:11.550]was published in 2005,
[01:11:13.331]people thought, "well, there's no need for this."
[01:11:15.877]"Why are we even doing this?"
[01:11:17.187]And it turns out...
[01:11:19.740]It turns out, yeah, we needed it quite badly.
[01:11:22.560]Yeah, no, thank you.
[01:11:24.577]"So if a student is interested in this area of research,
[01:11:29.370]non-viral gene delivery,
[01:11:31.230]what would you suggest they would need to do
[01:11:33.450]in order to be able to have adequate background
[01:11:36.210]to pursue this area?"
[01:11:37.710]That's a great question as well.
[01:11:39.450]You know, I think there's...
[01:11:41.160]Gene therapy or gene delivery
[01:11:43.714]is sort of a intersection of a variety of fields,
[01:11:47.880]from cell biology, molecular biology,
[01:11:49.593]genetics, biomedical engineering,
[01:11:51.694]chemical engineering, material science.
[01:11:54.341]I'm sure I'm not even remembering all the different fields.
[01:12:00.900]So I say that in that there's a variety of paths
[01:12:03.510]that one might take to get to working in this area.
[01:12:07.140]And if you're really interested in developing
[01:12:09.376]the materials around delivery,
[01:12:12.158]you might think about material science or engineering.
[01:12:14.940]If you're really interested in the genetics
[01:12:17.820]or the biology of it,
[01:12:19.470]then you may wanna pursue a degree
[01:12:21.120]in biochemistry or molecular genetics.
[01:12:24.960]Yeah, so there's a variety of paths
[01:12:26.760]because it's really a convergence of a variety of fields
[01:12:29.520]that came together to develop this field
[01:12:32.160]and to keep promoting it.
[01:12:34.080]Thank you, yeah.
[01:12:34.913]And do you actually...
[01:12:38.898]I know you have grad students.
[01:12:40.350]Do you have undergraduates and stuff-
[01:12:42.060]I do, yeah, yeah.
[01:12:43.200]So can you tell me what they do?
[01:12:45.330]Yeah, oh, they work on everything that I talked about.
[01:12:47.700]So they get to deliver genes and deliver small parts of RNA.
[01:12:52.574]We do a lot of that work
[01:12:53.940]that I didn't really talk about today.
[01:12:56.520]And they get to do...
[01:12:57.353]The undergrads, get to do all the assays.
[01:12:59.040]They typically don't get to do the animal works in the lab,
[01:13:01.050]but they get to do all the assays and development.
[01:13:03.180]And undergraduates in my lab all have their own project,
[01:13:06.270]working on some really important feature
[01:13:08.310]of maybe the delivery system
[01:13:09.780]or maybe an important feature of the DNA sequence.
[01:13:13.440]And I've trained almost 50 undergraduates in my lab
[01:13:17.010]since I've started here 15 years ago,
[01:13:19.050]so that's a really exciting part of my job,
[01:13:21.300]is to help undergraduates.
[01:13:23.610]Maybe they don't all fall in love with gene therapy,
[01:13:25.890]but most of them will fall in love with research
[01:13:27.720]and the idea of it and the excitement,
[01:13:30.510]the scientific inquiry, the process,
[01:13:33.300]how to run experiments,
[01:13:34.530]and so it's been fantastic.
[01:13:36.630]And we have two phenomenal undergraduates
[01:13:39.000]in the lab right now.
[01:13:40.384]Wow.
[01:13:41.217]Well, I think that's a wonderful place to wrap up.
[01:13:43.710]I wanna thank everybody online for joining us.
[01:13:46.515]Absolutely.
[01:13:47.348]And especially Dr. Pannier
[01:13:49.110]for giving us just an amazing lecture
[01:13:51.660]and so much to think about.
[01:13:53.130]Thank you so much.
[01:13:54.000]So, everybody, thank you very much.
[01:13:55.548]Thank you. (upbeat music)

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gene therapy
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Angie Pannier
Nebraska Lecture

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Nebraska Lecture with Angie Pannier:

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