Manipulating Biology: Costs, Benefits and Controversies

Background

In order to understand the direction of this unit, it is important to visit several topics.  First, we will examine environmental influences on genes and some mental disorders that are believed to have genetic links.  Next, we will look at the possible pathways to developing a therapy, and the mechanisms by which those therapies work for treating mental disorders at the genetic level.  Finally, I will address concepts that could hinder the advancement of this technology and its potential pitfalls.  Throughout this section of the curriculum unit, I will go back and forth by comparing what scientists are doing in our universe with what writers between DC Entertainment and Warner Bros. Television have scripted in the Black Lightning television series.  This television adaptation is essential to generating student interest in this unit.

Epigenetics:  Environmental Influence on Genes

The Black Lightning television series offers such a great platform to discuss genetic experimentation, but where do we begin?  It begins with answering this question:  What factors motivate or trigger aggressive behavior in the African Americans as portrayed in the TV show?  The first thing that comes to mind is oppression from the police.  Next are other things that I have noticed either in my lifetime or in my students’ lives:  low socioeconomic status, poor living conditions, minimal access to quality healthcare, being raised by a single parent, being orphaned, etc.  This list can become quite extensive.

While we know that social and environmental factors can influence aggressive behavior, there may be underlying genetic causes for aggression in people.  Aggressive behavior has been linked to attention-deficit/hyperactivity disorder and schizophrenia.  There are even claims that the genes and neural circuits that activate aggression are conserved between species, meaning that the study of animal models should provide comparable evidence.¹  However, there is still complexity in comparing aggression research between different animals.  There are non-human types of aggression such as offensive-, defensive- and predatory-subtypes.  Human aggression can also be subcategorized as adolescent-onset (rebellious), reactive (impulsive), or instrumental (non-impulsive) aggression.²

There is suspicion that each psychiatric disorder may prevail in exhibiting one subtype of aggression over another.  Research scientists are currently looking to zebrafish as the model organism for studies on aggressive behaviors.³  Zebrafish have already been used to study the presence of impulsivity, stress and anxiety, and even social behaviors, indicating that zebrafish are suitable for studying the different subtypes of aggression.

Recent reports of schizophrenia have shown that people of African heritage have higher rates of this mental disorder.⁴  Also, of those individuals diagnosed with schizophrenia, people of African heritage showed more severe symptoms than Caucasians.⁵  In addition, differences in the quality and scope of hallucinations and delusions have been noted for people of differing ancestry.

In 2015, a group of scientists compared the symptoms of psychosis in schizophrenia, schizoaffective disorder, and bipolar disorder between African Americans and Caucasians.⁶  They confirmed that diagnosis of schizophrenia was more common and the diagnosis of bipolar schizoaffective disorders was less frequent in African Americans compared to Caucasians.  The same study revealed that African Americans expressed symptoms of hallucinations and delusions across the three categories of disease previously mentioned.  Even African Americans with mild psychosis tended to over-endorse hallucinations and under-endorse delusions compared to Caucasians.  Other symptoms did not exhibit racial bias, nor did racial bias in assessment of symptoms explain differences in the proportion of the symptoms between the two groups.

The aforementioned studies ought to make you question whether or not environments influence the manifestation of systems.  Epigenetics is the study of how environmental pressures and social stressors can induce biological pathways that affect the health of organisms.  Another simpler definition is this:  “biochemical modifications that alter genetic expression but do not alter the DNA sequence”.⁷  While epigenetics is more evident in plant and animal species that reproduce quickly, the search for epigenetic effects in humans is only beginning.⁸  Due to the fact that so many variables serve as environmental pressures, it is difficult to isolate a causative effect, leaving only correlative data.

The Search for Aggression Genes

Let’s return to the science fiction in the Black Lightning television series for a moment.  Even though epigenetics is not well understood in humans at the moment in the real world, scientists in the DC universe seem to have some understanding.  Their response to aggressive behavior in African Americans with a vaccine is quite profound.  This implies that their so-called “vaccine” is not a vaccine by traditional convention.  The television show does indicate that this vaccine contains genetic material, meaning that the brain was not simply medicated with a drug.  They found a way to deliver genetic material to brain cells:  viruses.  Let’s examine the genetic material related to schizophrenia currently being studied in real life.

While the possible genetic markers of aggression are still being examined, schizophrenia is a severe mental disorder.  Symptoms include hallucinations, delusions, cognitive deficits, and apathy.  While inheritance patterns of the disease are complex and not completely understood, heritability has been estimated around 73-90%.⁹

Now, let’s review a few things about the substances that ultimately create genes:  deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).  They are both composed of four bases connected by a phosphate backbone.  There are 5 nucleobases.  DNA and RNA share 3 bases:  cytosine (C), guanine (G), and adenine (A).  The two bases, C and G complement each other in pairs.  Adenine has two complementary bases, depending on whether or not it is paring with DNA or RNA.  If a strand of DNA is paired with another strand of DNA, then its complementary base is thymine (T).  If a strand of DNA is paired with one of RNA, then its complementary base is uracil (U). DNA codes for the genetic makeup of the organism, while RNA codes, decodes, and regulates the expression of genes.  RNA can be classified into subtypes, such as messenger RNA (mRNA), guide RNA (gRNA), micro-RNA (miRNA), and long non-coding RNA (lncRNA).

Intermolecular forces ultimately drive the helical structure of DNA beginning with the bases.¹⁰  The bases form hydrogen bonds, and their matching pair is based on the arrangement of the hydrogen bond donor and acceptor at the end of each base.  The hydrogen bond that forms between the base pairs nearly cancels out the dipoles, making the bond nonpolar.

The expression of schizophrenia has been linked to micro-RNA (miRNA) and long non-coding RNA (lncRNA).¹¹  Reports have shown that there are several miRNAs related to schizophrenia such as miR-132/121 that affect the neuronal maturation and plasticity.  miR-138/125b both affect dendritic spine size, while miR-137 affects spine maturity.  miR-132 affects spine density, size, and maturity.  Moreover, miR-132 influences dendritic branching and synaptic integration in newborn hippocampal neurons. 

Targeting Aggression and Related Disorders

Now that we’ve explored the possibility of genes coding for aggression and other mental disorders, we still have to figure out how a vaccination is capable of treating a mental disorder.  We are so used to vaccines being used to prevent the onset of diseases such as polio, measles, mumps, chicken pox, and influenza.  How could the scientists in Black Lightning actually conceive the idea of developing a vaccine to treat mental illness?

Vaccines work by supplying a host with a dead or weakened pathogen in order to generate an immune response in the host, later protecting it in the event of a true pathogen infection.  Viruses are also capable of crossing the blood-brain barrier, unlike other microbes.  Also, it is an immune cells job to fight viruses and generate an immune response, not the brain cell.  Returning to Black Lightning’s case, the vaccine could not have been a real vaccine by definition, but rather a form of virus therapy. 

Since viruses are carriers of genetic information (either DNA or RNA), it is very plausible to load drugs or other genetic materials into those viruses.  This is already being put into practice via the use of phages (bacteria-specific viruses).  Even though bacteria can develop resistance to phages, scientists have found ways to work around that.¹²  They are creating or discovering phages that bind to the virulence factors (typically protein structures) of bacteria to drive evolutionary trade-offs in the bacteria.  Because the phages are lethal to the bacteria, they exert selection pressure for the bacteria to evolve phage resistance while simultaneously reducing their virulence (a trade-off).  In some cases the result is increased sensitivity to chemical antibiotics.  If scientists are able to develop phages that safely treat bacteria infecting the human body, then a “harmless” version of a virus should pose no problem if harnessed to deliver drugs and other molecules to brain cells.  A group in Pasadena, CA has developed a “harmless” virus to meet that objective.¹³  A mouse variant of a virus has been tested in vivo and a human variant has only been tested in vitro.  These virus variants transduce both neurons and astrocytes more efficiently than prior variants.

CRISPR Technology: The Genetic Editor

So back in the DC Comics universe, the evil scientists trying to keep African Americans in oppression via vaccinations have finally figured out how to target the brain.  We still need to know what types of molecules these viruses are pressuring our precious brain cells to uptake.  The answer is quite complicated, but catchy:  clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease 9 (Cas9).  Sometimes, these two are referred to as a combined system called CRISPR/Cas9 gene drive system (CGD).

The History of CRISPR

While clustered regularly interspaced short palindromic repeats (CRISPR) is relatively new in its name, its discovery dates back to the 1980s.¹⁴  In the late 1980s, scientists were first observing the repeat sequences in pathogenic bacteria.  The advancement of genome sequencing technologies during the 1990s really accelerated the ability to observe these repeating sequences in much of the bacterial world, but scientists did not understand the purpose of these sequences.  It was not until 2002 when two groups consistently and repeatedly observed the occurrence of these repeats, coining the acronym ‘CRISPR’.  At that time, scientists began to realize that CRISPR could be used to separate repeating sequences or to even repair DNA.  The true function of CRISPR was revealed in 2005 when research groups observed that the origin of these random sequences of CRISPRs were from viruses and plasmids, indicating that CRISPR may be a defense system in prokaryotes.

As we approach 2007, using bacterial cultures and phages, scientists conducted experiments hypothesizing that bacteria acquire these genetic spacers as a means for achieving phage resistance.  As a result, several discoveries were made.  First, CRISPRs can uptake phage DNA during cellular infection.  Second, the contents of the CRISPR spacer determine whether or not the bacterium will gain, lose, or exchange resistances via addition, deletion, or transplantation.  Finally, the Cas9 sequence was officially recognized as the adaptive immune system of bacteria.¹⁵

The workings of the CRISPR/Cas9 system

The CRISPR/Cas9 system works using a Protospacer Adjacent Motif (PAM) binding to locate target sequences (Figure 1a).  The most common form of the Cas9 nuclease comes from Staphylococcus aureus (SaCas9) and Streptococcus pyogenes (SpCas9), two types of bacteria responsible for staph infections and strep throat, respectively.¹⁶  SpCas9 requires a PAM sequences of 5’-NGG-3’, where “N” is any nucleobase that is followed by two guanine (G) bases.¹⁷  The PAM interaction uses two arginine residues to bind to the major groove of the guanine bases, a lysine residue to bind to the minor groove, and serine residue to bind to the phosphate group in front of the “N” nucleobase.  This will cause the DNA to unzip, allowing the complementary strand of gRNA to take its position, and Cas9 begins to rewrite the DNA using the gRNA.  Two nucleases (HNH and RuvC) work to cleave the DNA specifically between the third and fourth nucleobase pairs (Figure 1b).

Figure 1a.  Guide RNA helps Cas9 locate PAM sequence NGG.

Figure 1b.  Two nucleases (HNH and RuvC) cleave the DNA and begin to pair it with the RNA

CRISPR Trials on Schizophrenia

So while studies are on their way for markers of aggression, let’s examine a disease related to aggression: schizophrenia.  This mental disorder has mainly been treated using medications, but medications typically have adverse side effects.  Patients, wanting to avoid the side effects, will come off of the medication and their disease is now left unregulated.  As an alternative, scientists are looking to investigate the use of the CRISPR/Cas9 system as a means of directly correcting the mutations in DNA genomes and ncRNA genes.  A group at Duke University has already used the system to treat a human disease, Duchenne muscular dystrophy, within a living mouse model by replacing mutations with substitutions.¹⁸

Using CRISPR, scientists have been able to edit the non-coding regions of genomic DNA in both human and animal cell lines, even in schizophrenia research.  So far, the best use of this technology in schizophrenia research is the use of two gRNAs, allowing for two specific cuts to be made in order to delete a larger fragment of DNA.¹⁹  This dual gRNA technique is favorable because schizophrenia involves multiple gene alterations of ncRNAs.

One of the main drawbacks of schizophrenia research has been the lack of translatable animal models to determine the cause.  The CRISPR/Cas9 system allows for opportunity to create these animal models, target specific genes, and ultimately determine if one or a combination of ncRNAs lead to the development and progression of schizophrenia.²⁰

Implications of CRISPR Technology

Although many are in awe about the idea of cutting, replacing, and repairing genes with this technology, I’m sure the scientists who developed the vaccine as portrayed in Black Lightning were hopeful as well.  Unfortunately, things did not go as planned.  They accidently altered the DNA of African Americans to the point that they developed superpowers.  So let’s look at some of the things that could go wrong with CGD technology in the real world.

A risk concerning CGD technology is the possibility of extra (nontarget) deletions or insertions in the genome.  Recently, a group discovered in their experiment a combination of deletions and insertions at 17 sites in the mouse genome.²¹  The average deletion size was 9 base pairs, but some deletions approached 600 base pairs of DNA.  The group observed that during simultaneous targeting of loci, sequences were often removed between loci separated by large distances.  The same group concluded that the safest way to delete genes for now is through a two-step targeting mechanism.

While scientists have been optimistic about the discovery and use of CGD, there have already been reports of its resistance.  One would like to believe that nearly all fruit flies readily accept CGDs, but a strain of fruit flies from Tasmania have a resistance rate as high as 56 percent.²²  Scientists have proposed CGD as a way to exterminate invasive species, but varying resistance means that CGD efforts may not prevail in that context.  On the other hand, it has been reported that CGD systems are too invasive, meaning that it only takes a few organisms with CGD to invade a local population and then infiltrate neighboring populations.²³  Rather than try to control invasive species, scientists have proposed that the best application of CGD may be to prevent the spread of diseases such as malaria in mosquitos.

One issue concerning the Cas9 nuclease is that the resistance of the nuclease itself.  Using blood donations, 79% of donors’ blood developed antibodies against SaCas9, while 65% of donors produced antibodies against SpCas9.  In addition, 46% of donors’ blood contained anti-SaCas9 T-cells, while results also suggest that anti-SpCas9 T-cells may even exist.  Another issue is that even our trusted model, the zebrafish, is not compatible with SpCas9.²⁴  After considering various strains of the fruit fly and bacteria, it is highly likely that scientists will have to search for a variety of Cas9 genes in order to minimize resistance in zebrafish and humans and devise a translatable model.  Another alternative is to engineer Cas9 genes.²⁵

While scientists have not found a solid method for using CRISPR in humans, these setbacks will not stop them from dreaming.  Whether we like it or not, this technology is becoming our future.  The backstory behind the DC Comics’ superhero, Black Lightning, will no longer be just science-fiction.  Our understanding of science is evolving rapidly.  With Black Lightning now having to protect his neighborhood and his family from a drug war, there are still some questions.  Will CRISPR be used similarly in real life to genetically enhance human beings?  Or will we accidently create superheroes similar to Black Lightning?  What if CRISPR fails, edits too may genes, and begins to rob people of the life that scientists actually intended to improve, such as through healing of disease?  These are all questions that my students will grapple with.