INTRODUCTION

Practitioners in all healthcare settings need to understand and respect gender diversity. Pharmacists and pharmacy technicians can play a pivotal role in gender-affirming care by using inclusive language and developing comprehensive knowledge of pharmacologic interventions. The scope of gender affirming care ranges from puberty suppression to gender-affirming surgeries to align physical appearance with gender identity. Gender dysphoria is a very serious condition among transgender individuals that can lead to depression and suicide. Gender dysphoria is not solely internally driven and can be exacerbated by external stressors/stigma. This includes external aspects of experience such as distress associated with misgendering and social norms, social isolation, transphobia, etc.^1,2 It may be treated with hormone therapy and can require lifelong treatment. Healthcare professionals in the pharmacy setting can help these patients by creating a supportive environment that promotes adherence to their treatment. The SIDEBAR provides definitions for frequently used terms in transgender care.

SIDEBAR: Terminology^1-6

• Gender expression: External manifestations of gender, expressed through one’s behavior, body characteristics, clothing, haircut, name, pronouns, or voice. Typically, transgender people seek to make their gender expression align with their gender identity, rather than their assigned gender.
• Gender role: Behaviors, attitudes, and personality traits that a society (in a given culture and historical period) assigns as masculine or feminine and/or that society associates with or considers typical of the social role of men or women
• Gender identity/experienced gender: One’s internal, deeply held sense of gender. For transgender people, gender identity does not match their sex assigned at birth. Most people have a gender identity of male or female. For some people, gender identity does not fit neatly into one of those two choices (e.g., nonbinary). Unlike gender expression, gender identity is invisible to others.
• Sex: The best-known attributes include sex-determining genes, sex chromosomes, gonads, sex hormones, internal and external genitalia, and secondary sex characteristics.
• Sex assigned at birth: Sex assigned at birth, usually based on genital anatomy. AFAB refers to "assigned female at birth," and AMAB to "assigned male at birth."
• Transgender (also TGD, trans): An umbrella term for people whose gender identity and/or gender expression differs from what is typically associated with their sex assigned at birth. Not all transgender individuals seek treatment.
• Cisgender: A term for people whose gender identity and/or gender expression is aligned with what is typically associated with their sex assigned at birth.
• Gender dysphoria: The distress experienced when one’s gender identity and assigned gender are incongruent, often worsened by external factors like misgendering, social norms, isolation, and transphobia. Not all transgender people experience gender dysphoria; being transgender on its own is not a medical condition.
• Transition: The process during which transgender people change their physical, social, and/or legal characteristics consistent with their affirmed gender identity. Transgender individuals may initially transition socially and postpone medical transition. This is especially true for prepubertal children.
• Transgender male (also: trans man, female-to-male): Individuals assigned female at birth but who identify and live as men.
• Transgender woman (also: trans woman, male-to female, transgender female): Individuals assigned male at birth but who identify and live as women.
• Non-binary: An adjective describing a person who does not identify exclusively as a male or a female. Non-binary people may identify as being both male and female, somewhere in between, or as falling completely outside these categories. While many also identify as transgender, not all non-binary people do.
• Embodiment goals: Body qualities/characteristics that a patient wishes to have (e.g. shape, feeling, behavior of body).

Guidelines

The World Professional Association for Transgender Health (WPATH) publishes the Standards of Care for the Health of Transgender and Gender Diverse People (SOC),⁷ which is the main guideline used internationally for transgender health. This continuing education activity is based on SOC Version 8.

The Endocrine Society develops the Endocrine Treatment of Gender-Dysphoric/Gender-Incongruent Persons: An Endocrine Society Clinical Practice Guideline.³ These guidelines establish a framework for the appropriate treatment of individuals and standardize terminology to be used by healthcare professionals. This activity cites these guidelines as well.

Inclusive Language

Everyday language often revolves around the concept of two genders and one sexuality, potentially overlooking diverse identities. Using gender-inclusive language demonstrates respect and acknowledgment of all gender identities while eliminating assumptions. Using they/them pronouns instead of he/his or she/her when unsure of someone's pronouns is best.⁶ Healthcare professionals can demonstrate inclusivity by introducing themselves with their own pronouns and asking patients how they prefer to be addressed. Common pronouns include she/her/hers, he/him/his, and they/them/theirs..⁶ Healthcare professionals should avoid stating that a person is transgender to others unless that is how the person identifies and is comfortable with sharing that information.⁶ Another suggestion is saying “feel free to bring your spouse or partner” instead of “feel free to bring your husband or wife.”⁶

PAUSE AND PONDER: A fellow colleague consistently misgenders a transgender patient despite being corrected. How would you address this behavior while promoting inclusivity and respect in the pharmacy setting?

Physiology

Puberty is the process of an adolescent becoming capable of reproduction. This process involves the hypothalamic-pituitary-gonadal axis. The first biologic sign of puberty is an increase in endogenous gonadotropin hormone-releasing hormone (GnRH).⁷ GnRH produced by the hypothalamus causes the pituitary gland to secrete gonadotropins: luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH and FSH stimulate gonads to produce sex hormones.⁸ Table 1 compares puberty in individuals with ovaries and those with testes.

Table 1. Patterns of Typical Puberty⁷

ADULTS

Feminizing Pharmacology

The goals of feminizing pharmacology care are the development of female secondary sex characteristics and the minimization of male secondary sex characteristics. Treatments include externally dosed estrogens and antiandrogens. The selection of therapy should include a patient-specific evaluation of patient goals, medical conditions, medication risk/benefit, and economics.⁷

Estrogen promotes feminine features such as breast growth. Antiandrogens diminish masculine features by reducing endogenous testosterone. Examples of antiandrogens include spironolactone, cyproterone acetate, GnRH agonists, and 5-alpha reductase inhibitors.

Estrogen

In gender-affirming care, estrogen (or 17-beta estradiol) used for feminizing treatment is chemically identical to estrogen produced by human ovaries.^3,7 Multiple dosing formulations exist including oral tablets, topical patches, intramuscular injections, and topical gels/sprays. A major safety concern for anyone using exogenous estrogen is the increased risk of thromboembolic events. 17-beta estradiol is generally preferred over synthetic estrogens for the ease of monitoring serum levels and its lower risk of thromboembolic events. For those older than 45 years and those at a higher risk of venous thromboembolism (VTE), the guidelines recommend transdermal formulations. Cardiovascular screening is essential to identify patients at higher VTE risk. Due to multiple Boxed Warnings in the product labeling and potentially significant adverse events, providers should use caution and consider the risks and benefits of estrogen therapy.^3,7

The desired effects of exogenous estrogen include breast development, redistribution of facial and subcutaneous fat, reduction of muscle mass, reduction of body hair, and reversal of scalp hair loss. Exogenous estrogen will not alter voice or height in adults. Table 2 describes available estrogens used in gender-affirming care.^3,7

Table 2. Estradiol for GAHT⁹

Antiandrogens

Antiandrogens, listed in Table 3, can reduce endogenous testosterone levels or its activity and are usually used in combination with estrogen for feminizing effects. While antiandrogens may be employed as monotherapy, combination treatment can help minimize the amount of estrogen necessary for gender-affirming effects and therefore reduce the estrogen-associated risks. General adverse effects of antiandrogen monotherapy may include hot flashes, low mood or energy, and bone loss when used long term.⁷

Table 3. Antiandrogens for GAHT^10-13

Spironolactone is a potassium-sparing diuretic that has many therapeutic uses including in heart failure and hypertension. Spironolactone is also an aldosterone receptor antagonist that can cause positive effects, such as gynecomastia (an increase in breast gland tissue) for transgender women. Gynecomastia is dose and duration dependent, occurring in approximately 10% of patients. The diuretic and antihypertensive effects may become apparent within two to three weeks potentially resulting in excess urination and low blood pressure.^3,14

GnRH agonists (histrelin, leuprolide) work by continuously activating the pituitary gland to make more LH and FSH. This overactivation eventually suppresses sex steroid production. Out of pocket costs for these medications can range anywhere from $10,000 to $50,000 for three months, thereby significantly limiting their use.¹⁶

WPATH does not regularly recommend other drugs like 5-alpha reductase inhibitors, finasteride or dutasteride, as data supporting use in the transgender population is sparse.⁷

Cyproterone acetate is a synthetic progestin that inhibits testosterone synthesis and action. This medication is not approved in the United States but may be used elsewhere.³

PAUSE AND PONDER: A transgender patient presents with concerns about the different formulations of estrogen available for feminizing treatment. How would you explain the advantages and disadvantages of each formulation?

Masculinizing Pharmacology

The goals of masculinizing treatment are the development of male secondary sex characteristics and the minimization of female secondary sex characteristics. Masculinizing GAHT typically consists of testosterone.

Testosterone

Testosterone treatment in transgender patients is used in the same way that it is used for hypogonadism in cisgender male patients. Table 4 illustrates the various testosterone formulations in transgender care, including intramuscular, transdermal, and implantable options. Intramuscular injections may be preferred due to available clinical data, efficacy, patient satisfaction, and low levels of injection pain and local irritation relative to subcutaneous injections. Prescribers and patients should not interchange products without considering pharmacodynamic differences.^7,16

Positive effects of masculinizing hormones include irreversible deepening of voice, increased body/facial hair, and increased lean muscle mass. Patients and care teams should know that masculine hormone therapy will not reverse previous feminine features such as breast tissue. Achieving physiological testosterone levels will usually suppress menses. If testosterone monotherapy doesn’t suppress menses, additional therapies can be used including progestins, GnRH agonists, and aromatase inhibitors. Patients may continue testosterone therapy for life unless medically contraindicated.¹⁶

Table 4. Testosterone Formulations for GAHT ¹⁷

CHILDREN

GAHT is not recommended in children who have not begun endogenous puberty. Healthcare providers should take a non-pharmacologic approach instead for pre-pubertal individuals, including psychosocial gender-affirming care. Patients should continue gender exploration regardless of social transition.⁷

ADOLESCENTS

GAHT’s goal in adolescents is pubertal suppression. Halting progression of puberty allows adolescents to explore gender identity and embodiment goals. Although suppression is reversible, the treatment team should discuss fertility preservation before starting supression.⁷ Current timing and readiness estimates are as follows: pubertal suppression at 12 years old, GAHT at 16 years old, and surgery at 18 years old.

Pubertal Suppression Pharmacology

Healthcare providers employ Tanner staging to track children’s development during puberty. It outlines five specific stages for the physical changes during this period, including genital, breast, and pubic hair development. Patients should reach Tanner Stage 2 before initiating pubertal suppression. In Tanner Stage 2, individuals who are assigned female at birth would experience breast budding while those who are assigned male at birth would experience external genitalia enlargement.⁶ Menstrual suppression is recommended for patients assigned female at birth with gender incongruence, regardless of testosterone therapy.⁷

Pharmacologic Agents

Gonadotropin-Releasing Hormone (GnRH) Agonists

GnRH agonists cause partial regression of secondary sex characteristics and inhibit physical functions, such as menses and erections, by decreasing concentrations of gonadotropin and sex steroid hormones. Supraphysiological doses of sex steroids are not needed with GnRH agonists.⁷

Short-term hypertension is an important counseling point. Individuals who are older than 14 years also have a long-term risk of poor bone health because of the lack of exposure to adequate levels of sex steroid hormone levels. Sex steroid hormone therapy, however, induces rapid recovery of bone mineralization rate.⁶

For feminizing therapy, patients should continue puberty blocking until gonadectomy. Masculinizing pharmacology is covered in the following Hormone Therapy section. If patients have functioning uterus and ovaries, health care providers should counsel on potential breakthrough menstrual bleeding two to three weeks after GnRH agonist initiation.

Progestins

Oral or injection depot progestins are recommended if GnRH agonists are unavailable or cost-prohibitive.⁷

Patients can also receive these progestins if they seek menstrual suppression only⁷:

• Oral progestin-only pills (contraceptive and non-contraceptive options)
• Medroxyprogesterone injection (e.g. Depo-Provera)
• Levonorgestrel intrauterine device (e.g. Mirena, Liletta, Kyleena, Skyla)
• Etonogestrel implant (e.g. Nexplanon)

If a patient continues to have menstrual bleeding after taking progestin and/or is seeking a contraceptive, healthcare providers can consider a combination progestin-estrogen product for amenorrhea and counsel on possible breast development. Combined formulations include oral contraceptive pills, transdermal patches, and vaginal rings. Note that increased breakthrough bleeding is associated with lower dose ethinyl estradiol in combined oral contraceptives.⁷

Hormone Therapy (HT)

The patient’s health care professionals will need to measure hormone levels during gender-affirming treatment. The purpose is to ensure that endogenous sex steroids are falling and the sex steroids administered to the patient stay at appropriate levels. Appropriate levels are determined by (1) the patient’s treatment goals and (2) the patient’s Tanner stage. Timing of pubertal suppression determines sex steroid HT regimen, as shown in Table 5.⁷

Table 5. Sex Steroid Hormone Therapy Regimen Based on GnRH Agonist Treatment Timing⁷

WPATH suggests 12 months as sufficient time for psychological adaptations to physical changes due to GAHT. Healthcare providers should monitor sex steroid levels every three months during the first year of HT or with dose changes. Once the prescriber titrates the patient’s dose to the adult maintenance dose, monitoring one to two times annually is sufficient. As GAHT can activate the hypothalamic-pituitary-gonadal axis, prescribers may need to add a GnRH agonist as adjunctive therapy after GAHT initiation to avoid development of characteristics associated with the patient’s sex assigned at birth.⁷

For masculinizing GAHT, testosterone monotherapy at physiologic doses is typically sufficient to decrease estrogen secretion by the ovaries. Injection, transdermal, and subcutaneous pellets are available androgen formulations. Key counseling points include the possibility of developing androgenic acne or sexual dysfunction.

Patients on GnRH agonists should continue them until they reach maintenance testosterone level. If the patient was not on a GnRH agonist as an adolescent, then no concomitant GnRH agonist is needed. While testosterone usually suppresses menstruation in the first six months of therapy, healthcare providers should make sure to counsel adolescents on possible pregnancy despite amenorrhea because they can still ovulate.⁷

NON-PHARMACOLOGIC INTERVENTIONS

Chest Binders

Chest binding refers to compressing breast tissue to achieve a flatter chest appearance. Research indicates that up to 87% of individuals identifying as transgender males have tried chest binding.⁶ Various methods used for binding include commercial binders, sports bras, layering clothes, elastics, and bandages.⁷

Healthcare providers play a crucial role by making patients aware of chest binding’s potential advantages and risks. Transgender men who bind often report benefits such as increased comfort, enhanced safety, and reduced instances of misgendering.⁷

Negative physical impacts of chest binding include back or chest pain, difficulty breathing, and feeling overheated.⁷ Severe health issues like skin or respiratory infections and rib fractures, which are rare, have been linked to adult chest binding.⁶ Providers can lower the risk by counseling patients on safe binding methods, like binders specifically designed for gender-diverse individuals, to lessen the likelihood of serious health complications. Meanwhile, patients should avoid unsafe methods like duct tape, ace wraps, or plastic wrap due to their potential to constrict blood flow, harm the skin, and restrict breathing.⁶ If negative health effects occur, seeking guidance from medical professionals experienced with transgender and gender-diverse patients is sensible.⁷

Tucking

Genital tucking involves positioning the penis and testes to minimize the visibility of genital bulges.⁷ This can be done by tucking the penis and testes between the legs or placing the testes inside the inguinal canal while pulling the penis back between the legs.⁷ Underwear or specialized garments called gaffs are commonly used to keep the genitals in place.⁷

Research on the risks and advantages of tucking in adults is limited, and no studies conducted specifically address youth.⁶ Previous research has highlighted that snug undergarments might lead to reduced sperm concentration and motility. Moreover, higher scrotal temperatures due to tucking might theoretically impact sperm production and fertility.⁷ However, no conclusive evidence confirms these negative effects. More data is needed to understand the risks and benefits of tucking.

Voice Therapy

Hormone treatment for transgender and gender diverse (TGD) individuals has potential effects on voice and communication. While estrogen treatment does not usually cause measurable voice changes, testosterone treatment can lead to desired shifts in voice pitch and male attributions but may also result in undesired outcomes.⁷

Research indicates that some TGD individuals experience positive effects from testosterone, such as lowered voice pitch and increased satisfaction, aligning with their gender identity. However, a significant portion may face challenges like insufficient pitch changes, vocal quality issues, limitations in singing range, and vocal instability post-treatment.^7,18 A meta-analysis examined about 600 patients across 19 studies to determine the effects of at least one year of testosterone therapy. The patients were all at least 18 years old, with an average age of 35 years. According to the results, approximately 21% of participants did not reach the expected normative frequencies associated with cisgender males. Additionally, a similar percentage reported incomplete alignment between their voice and gender identity, experiencing voice-related challenges or incongruence. Furthermore, 16% of individuals undergoing testosterone therapy expressed dissatisfaction with the changes in their voice.¹⁸

It's essential to provide accurate counseling beforehand to establish realistic expectations and avoid potential disappointment. Referral to voice and communication specialists can address concerns through tailored voice training and assisting those dissatisfied with outcomes or lacking access to hormone treatment.⁷

Electrolysis And Laser Hair Removal

Hair removal is a significant part of transitioning for many transgender women (or females), particularly for those seeking a more stereotypical feminine appearance. The process helps alleviate dysphoria, boosts self-confidence, and enhances overall quality of life.¹⁹ The U,S, Food and Drug Administration recognizes laser hair removal as a permanent hair removal method.¹⁷ It targets melanin in hair follicles using laser light waves.¹⁹ An alternative, electrolysis, also removes hair permanently using a small probe inserted into individual hair follicles to administer an electric current.¹⁷

For individuals undergoing GAHT, healthcare providers need to keep specific considerations in mind. Among transgender women taking estrogen¹⁹

• Estrogen hormone therapy often results in an overall reduction in body hair, except for minimal effects on facial and genital hair.
• The extent of hair removal (complete hairlessness or retaining some hair) depends on personal preference and comfort levels.
• Many transgender women find facial and neck hair removal crucial for increased self-confidence, reduced dysphoria, and a sense of safety in public spaces.
• Those prioritizing gender-affirming lower surgeries like vaginoplasty (vagina construction surgery) require completely hair-free areas for surgery preparation, as hair growth within transformed tissue can cause complications.

For transgender men taking testosterone¹⁹

• Testosterone hormone therapy generally leads to increased body hair growth.
• Preparing for surgeries like phalloplasty (penis construction surgery) often requires completely hair-free skin areas for successful procedures, the specific area depending on the type of surgery planned.
• It is important to note that electrolysis doesn't prevent the growth of new hair follicles activated by testosterone. Therefore, new hair growth might occur in areas previously treated with electrolysis due to testosterone’s effects on stimulating hair follicles.

The adverse effects of hormone therapy may be beneficial for some patients, while being detrimental to others. It is critical for healthcare providers to educate patients on hair removal methods and their impact within the context of hormone therapy, so that patients understand what to expect.

Gender-Affirming Surgery

Gender-affirming surgery is often a crucial milestone for many, but not all transgender adults.³ Broadly classified into two categories, surgeries (1) directly impact fertility or (2) have no effect on fertility.³ Surgeries that alter fertility involve procedures like the removal of male genitalia (penis and gonads) for transgender women or the removal of female reproductive organs (uterus and ovaries) for transgender men.³ Surgeries that do not affect fertility include chest masculinization or breast augmentation, facial feminization, or facial masculinization surgeries.³

There is a lack of evidence supporting the routine discontinuation of hormone therapy before planned surgeries.²⁰ The majority of evidence advocating for estrogen cessation before surgery is derived from studies involving oral synthetic estrogen regimens (ethinyl estradiol), which are uncommon in transgender patients.¹⁹ WPATH advises maintaining estrogen therapy both before and after surgical procedures in transgender women, particularly in those without specific risk factors such as smoking, family history of VTE, or the use of synthetic estrogens.⁷ If the patient has the previously mentioned risk factors, the prescribing endocrinologist should discuss estrogen therapy cessation in the perioperative setting openly with the patient and make decisions collaboratively.²⁰ Typically, an acceptable timeframe for estrogen discontinuation is two to four weeks before the procedure.²⁰ Anticipated physiological and psychological withdrawal symptoms may include anxiety, autonomic hyperactivity, depression, decreased seizure threshold, and fatigue.²⁰ This comprehensive approach ensures a thoughtful consideration of hormonal factors in the surgical process for transgender women.

Evidence suggests that there is generally no need to discontinue testosterone treatment routinely in transgender men before scheduled or elective surgery despite the concern that testosterone can be aromatized to estradiol, which theoretically could increase the clotting risk.²⁰ A recent systematic review found no association of increased VTE complications after surgery with perioperative testosterone use.^21,22

The decision to undergo these surgeries is deeply personal and varies based on individual needs, preferences, financial access, and dysphoria. Access to comprehensive information, counseling, and support from healthcare professionals helps patients make informed decisions aligned with their gender identity and long-term goals while understanding the potential impact on fertility.³

PAUSE AND PONDER: A transgender patient would like to undergo breast augmentation but has only been on hormone therapy for six months. Is she eligible for the surgery?

Chest or “Top” Surgery

“Top” surgery is either removing breast tissue for a more masculine appearance or enhancing breast size and shape for a more feminine appearance. Breast surgery is a type of gender-affirming surgery with no impact on fertility.³ Since breast size varies among females, it is advisable for transgender women to postpone breast augmentation surgery until they have undergone at least two years of estrogen therapy.³ This is recommended because the breasts continue to grow during this period of hormone therapy.³

Meanwhile, the primary masculinizing surgery for transgender men is a mastectomy, which also has no impact on fertility.³ While breast size only partially regresses with androgen therapy, discussions about mastectomy in adults typically occur after androgen therapy starts.³ However, in some cases where trans-masculine adolescents present after significant breast development, a mastectomy may be considered approximately two years after starting androgen therapy and before the age of 18.³ Treatment decisions should be individualized based on the individual’s physical and mental health status. ³

Genital or “Bottom” Surgery

Genital or “bottom” surgery involves transforming and reconstructing the genitalia. According to The Endocrine Society Clinical Practice Guideline Criteria for Gender-Affirming Surgery, the following factors may influence decisions related to fertility preservation³:

1. Persistent and well-documented gender dysphoria
2. Meets the legal age requirement in the relevant country
3. Use of gender-affirming hormones for a continuous 12-month period, unless there is a medical contraindication
4. Successful in living full-time as a new gender role for the duration of 12 months
5. Well-controlled management of any significant medical or mental health conditions, if present
6. Demonstrated knowledge of practical aspects related to surgery, including cost, required lengths of hospitalizations, likely complications, and postsurgical rehabilitation.

Gender-affirming surgeries for transgender women that affect fertility include procedures like gonadectomy (orchiectomy), penectomy, and the creation of a neovagina.³ In the case of transgender men, surgeries that affect fertility include an oophorectomy, vaginectomy, complete hysterectomy, and the creation of a neopenis.³ Infertility can occur from both the temporary consequences of gender-affirming hormone therapy and permanent effects of gender-affirming surgeries (GAS).⁶ It is crucial to engage in ongoing discussions about infertility risks and fertility preservation options with transgender individuals and their families before and after initiation of therapies and surgeries.⁶

OTHER CARE IMPACTED BY GAHT

Anticoagulation

When assessing the risk of VTE associated with GAHT, it is crucial to consider the alternative—the risk of not providing GAHT.²² Withholding GAHT could lead to adverse mental health consequences that might outweigh the risk of VTE.²⁰ Some individuals may seek hormone therapy outside clinical care settings (that is, from unreliable sources) if healthcare providers refuse to provide it.²²

For transgender individuals experiencing VTE while on GAHT, treatment should align with current therapeutic anticoagulation recommendations for cisgender individuals.²⁰ The American Society of Hematology guidelines recommend direct oral anticoagulants (DOACs) over other options due to a lower risk of bleeding.²² Despite limited data in transgender people, recent case reports suggest DOACs are effective in those with VTE during GAHT. In severe cases threatening limbs, clinicians can consider thrombolysis and recommend it as they would in cisgender individuals.²² GAHT discontinuation during an acute VTE episode is often recommended, especially with above normal hormone levels, but this recommendation lacks extensive data.²² Clinicians and patients should discuss the potential risks and benefits of continuing GAHT during an episode.

Following acute VTE treatment, the decision to resume GAHT becomes complex. Many clinicians continue GAHT, even in patients with previous VTE, along with full-intensity anticoagulation to prevent recurrence.²² Although researchers have not conducted systematic evaluation in transgender patients, this approach was effective in cisgender women on hormone therapy.²² While cisgender patients may reduce anticoagulation dose after initial therapy, it is uncertain if dose reduction is prudent with GAHT. Shared decision-making, emphasizing the risks and benefits, remains crucial in determining the course of GAHT post-VTE treatment.

Estimating Kidney Function

Many healthcare providers are accustomed to using the Cockcroft-Gault equation to estimate kidney function.²³ This formula factors in sex, causing uncertainty as to how to calculate creatinine clearance in transgender patients. Although GAHT affects muscle mass and creatinine, using the duration of therapy is unreliable because the timing and magnitude of effect differ by individual. As such, cystatin C is often more accurate than creatinine. Acknowledging that cystatin C levels are not accessible at all practice sites, some experts suggest using the Cockcroft-Gault-calculated male and female creatinine clearance estimates as a range. Other methods include using vancomycin clearance calculations.²³

Fertility Preservation

Individuals who have transitioned may wish to have biological children for personal reasons, to maintain a genetic connection with their offspring, or to conform with cultural expectations.²² While many transgender individuals may not initially consider having children, their desires can change over time.²⁴ In a survey of 50 transgender men who had undergone gender-affirmation surgery, the majority (77%) had not contemplated fertility preservation before GAHT. An average of 9.9 years after starting testosterone therapy, 54% of participants wanted to have children.²⁵ The study also found that participants with children had a higher quality of life than those who did not have children.²⁵

Fertility preservation through the cryopreservation of sperm and oocytes is a well-established technique, available for pubertal, late pubertal, and adult individuals assigned male or female at birth^.6 Ideally, clinicians should present this option before initiating GAHT.⁷ Clinicians can offer embryo cryopreservation to adult transgender individuals, particularly those who have completed puberty, express a desire to have a child, and have a willing partner.⁷

While research has shown semen parameters may be compromised after the initiation of GAHT, a small study indicated that when treatment was discontinued, semen parameters were minimally altered.²⁶ In regards to oocyte preservation, there is no expectation that assisted reproductive technology (ART) treatments would be different for TGD patients compared to cisgender patients.⁷ The only potential variations are individuals with confounding factors related to infertility.⁷

Barriers to fertility preservation include financial constraints, invasiveness of procedures, and concerns about mistreatment or bias from healthcare providers.²⁴ Some fertility preservation procedures, such as sperm or egg collection, can serve as reminders of their sex assigned at birth and may trigger gender dysphoria.²⁴ Transgender individuals who pursued ART reported mixed experiences. Some have positive encounters with supportive providers who use gender-neutral language. Others experienced misgendering in clinical documentation.²⁴ Patients should be provided with options for future family planning whether they desire biological children or not.

BARRIERS IN PRACTICE

Laws and Legislation

When the Supreme Court tackled the Conservative challenge to the Affordable Care Act (ACA), the focus was on whether it was constitutional to mandate health insurance purchase.²⁷ This overshadowed Section 1557's anti-discrimination provisions protecting transgender individuals. The 2016 Rule, finalized by Health and Human Services, extended Title IX's anti-sex discrimination coverage to include "gender identity," ensuring equitable access to healthcare for transgender patients. The Trump administration attempted to reverse these protections but faced legal obstacles. This led to the Supreme Court's ruling in Bostock v. Clayton County, which affirmed protections for transgender individuals under existing civil rights statutes. Despite the Trump administration's efforts, subsequent legal challenges and judicial actions further emphasized protections for transgender individuals, leading to ongoing debates and policy shifts under the Biden administration.²⁷

The Bostock ruling, initially about employment, now impacts transgender minors' access to gender-affirming care.²⁷ Federal courts often interpret Title VII and Title IX together, meaning they see both statutes as prohibiting sex-based discrimination similarly.²⁵ This means that the Biden Administration can confidently enforce protections for gender identity under Title IX, following Bostock's reasoning.²⁷ Since Bostock's ruling was made under a majority conservative Supreme Court, it's unlikely to be reversed. This leaves opponents of gender-affirming care to explore other tactics, like raising religious objections or labeling such care as "experimental" or "medically dangerous."²⁷ These viewpoints circumvent many physicians’ clinical judgement, thus restricting how they practice medicine.

Over the past 18 months, the number of states implementing laws that restrict or prohibit minors' access to gender affirming care has increased dramatically.²⁸ Currently, the count of states with restrictions increased from just four states in June 2022 to 23 states by January 2024. Among these states, 17 have fully enacted their restrictions, while four face temporary injunctions, and one is permanently blocked pending appeal. The laws vary in complexity, with some focusing on specific aspects of gender affirming care, such as GAHT or gender affirming surgery. Additionally, while these laws primarily target minors' access to care, they often include provisions affecting other groups, such as parents, medical providers, and teachers.²⁸ These laws and policies cause potential harm to the wellbeing of young TGD, thus emphasizing the need for continued advocacy and support for inclusive and affirming practices.

Insurance Coverage

Ensuring comprehensive healthcare for transgender individuals requires access to both gender-affirming care and a wide range of inclusive services. However, numerous barriers have historically hindered access, with lack of health insurance coverage being shaped by intersecting factors at the individual, interpersonal, and structural levels.

Many state Medicaid programs exclude coverage for gender-affirming care, posing a significant concern given the elevated prevalence of poverty among transgender people. In a study utilizing Behavioral Risk Factor Surveillance System (BRFSS) data, gender non-conforming individuals were nearly twice as likely as cisgender women to report unmet care needs due to financial issues.²⁹According to the 2015 United States Transgender Survey, 25% of insured respondents encountered insurance discrimination.²⁹ Experiences included denial of coverage for gender-specific services, such as cancer screenings (13%), for care unrelated to gender affirmation (7%), and for gender-affirming surgery (55%) or hormone therapy (25%).²⁹ TGD individuals (33%) reported negative experiences related to being transgender when seeing a healthcare provider in the previous year, which includes verbal harassment, physical assault, or treatment refusal.²⁹ Additionally, 23% refrained from seeking care when needed in the previous year due to fear of mistreatment.²⁹

A 2019 systematic review revealed that 27% (range, 19% to 40%) of transgender individuals reported outright denial of healthcare.²³⁰ Assessments of provider readiness indicate that many clinicians lack training in transgender clinical and cultural competency, potentially contributing to interpersonal discrimination in healthcare settings.

Assessing Implicit Bias, Addressing Bad Behavior

1. Respect Gender Identity: TGD individuals deserve the same respect as cisgender patients. Address patients by their chosen name and pronouns, reflecting their gender identity. Avoid gossiping or making jokes about patients and treat them with the same respect you would want at work.³¹

2. Recognize Workplace Values: Understand the difference between your personal beliefs and the workplace values of dignity and respect for everyone. While you are entitled to your opinions, professionalism requires setting aside personal views and treating everyone with fairness and courtesy in the workplace.³¹

3.Seek Feedback and Learn from Mistakes: Don't be afraid to ask for feedback from TGD individuals about how you can better support them or avoid inadvertently causing harm. Be open to learning from your mistakes and apologize if you unintentionally offend someone. Additionally, take the initiative to stay updated on evolving terminology and practice recommendations, as ideas/norms about gender are fluid and change over time. Learning from reputable sources, such as The Trevor Project's Guide to Being an Ally to Transgender and Nonbinary Youth, shows a genuine commitment to fostering inclusivity and respect in the workplace.^31,32

4. Support Colleagues with Challenges: If you notice co-workers struggling to adjust to interactions with TGD individuals, offer gentle reminders about using the correct name and pronouns. Help them understand the impact of their behavior on both the TGD individual and the workplace environment. Encourage empathy and understanding to foster a more inclusive workplace culture.³¹

5. Intervene When Appropriate: If you witness blatant inappropriate behavior or discriminatory actions with no remorse towards a TGD individual in the workplace, don't remain silent. Find appropriate ways to intervene, whether it's addressing the behavior directly, reporting it to management, or offering support to the affected individual. Standing up against prejudice and discrimination helps create a safer and more inclusive work environment for everyone.³¹

CONCLUSION

Embracing gender diversity and providing gender-affirming care is essential in healthcare settings, including pharmacies. Pharmacists and pharmacy technicians who employ inclusive language and possess a thorough understanding of pharmacological interventions play a critical role for transgender individuals. Pharmacists can help tailor GAHT regimens to individual patient needs, promoting safety and helping patients achieve their goals towards gender affirmation. Pharmacists and pharmacy technicians can create a more inclusive and safer environment when interacting with patients. As healthcare workers in the pharmacy setting, we can provide patients with the resources they need without judgement.

Download CE Activity

INTRODUCTION

Gene therapy is a medical approach to replace missing genes or correct dysfunctional genes in patients with genetic diseases. Gene therapies are best suited for patients who have well-defined, single (monogenic) mutations.^1,2 Many are rare diseases for which there are few, if any, treatment options. Therapeutics to correct gene mutations are biologics with the potential to cure a variety of previously incurable diseases such as muscular dystrophy, hemophilia, cystic fibrosis, and some types of blindness. Scientists are also developing gene therapies to stop cancer-causing genes and boost immune responses against tumors.

The term “gene therapy” encompasses multiple broad strategies that can be separated into two general approaches: in vivo (performed inside an organism) and ex vivo (cells that are removed from the organism, genetically modified, and then returned to organism).³ Within these approaches, multiple techniques and delivery systems are currently used or in development:^4,5

• Viral vectors (in vivo or ex vivo)
• Non-viral delivery particles (in vivo or ex vivo)
• Gene editing (in vivo or ex vivo)
• Cell engineering (ex vivo only)

The entire field of gene therapy is beyond the scope of this activity. This activity’s focus will be in vivo gene delivery via engineered adeno-associated virus (AAV), with some discussion of gene therapy’s basics and background. This is valuable information for pharmacists and pharmacy technicians as this complex biologic class matures and more gene therapies become available to patients.

BASICS OF GENE THERAPY

Gene Therapy Terminology

People outside of the field may be unfamiliar with many terms used to describe gene therapies. Table 1 defines common terms.

Table 1. Common Terms Used to Describe Gene Therapy^6,7,8

DNA, deoxyribonucleic acid.

We have used brand drug names preferentially throughout this activity because generic names for gene therapies are complex (See Sidebar).

SIDEBAR: What’s in a Name? How Are Gene Therapies Named?^9,10

For in vivo gene therapies, the generic names are composed of two words:

• First word corresponds to the gene component
  • Prefix: random element to provide unique identification
  • Infix: element to denote the gene’s pharmacologic class (gene’s mechanism of action)
  • Suffix: element to indicate “gene”
• Second word corresponds to the vector component
  • Prefix: random element to provide unique identification
  • Infix: element to denote the viral vector family
  • Suffix: element to identify the vector type (non-replicating, replicating, plasmid)
  • 4-letter distinguishing suffix: devoid of meaning and attached to the core name with a hyphen

Example:

The Pros and Cons of Gene Therapy 

Patients and/or caregivers considering gene therapy should consider several factors^11-13:

• Duration of efficacy
  • PRO: Potential exists for permanent correction of a disease state. If successful, patients may not need lifelong treatments/medications.
  • CON: Gene therapy is relatively new and long-term efficacy data is lacking. If gene expression wanes over time, patients may need redosing (which may be impossible) or alternative treatments.
• Safety
  • PRO: Regulatory agencies have approved several products, and safety data is available.
  • CON: Complete understanding of adverse effect mechanisms is lacking, and long-term safety information doesn’t exist.
• Cost
  • PRO: A curative gene therapy may be more cost-effective than a lifetime of other medications/treatments.
  • CON: Available gene therapies are very expensive, $2.9 to $3.5 million for the AAV in vivo therapies approved in 2022 and 2023.

The History of Gene Therapy

Gene therapy is a relatively new field. Clinicians ran the first clinical trial in 1990. Scientists made several advancements in the 1990s and early 2000s, and progress has rapidly accelerated since then.¹⁴ (See Sidebar).

ABBREVIATIONS: AAV, adeno-associated virus vector; Ad, adenovirus; ADD, adenosine deaminase deficient; ALL, acute lymphoblastic leukemia; CALD, cerebral adrenoleukodystrophy; CAR T, chimeric antigen receptor; Cas9, CRISPR-associated protein 9; CRISPR, clustered regularly interspaced palindromic repeats; DMD, Duchenne muscular dystrophy; EMA, European Medicines Agency; FDA, Food and Drug Administration; HSC, human stem cells; LV, lentivirus; DLBCL, diffuse large B-cell lymphoma; SCD, sickle cell disease; SCID, severe combined immunodeficiency disorder; SMA, spinal muscular atrophy; TDT, transfusion-dependent thalassemia.

Gene Therapy Systems

Viral Vectors

Prototypical gene therapy is an in vivo approach to curing genetic diseases. It uses a delivery vehicle, such as an engineered virus that encapsulates the gene of interest (GOI) for injection into a patient by various routes, such as intravenous (IV), intraocular, or intrathecal.³⁹ This method takes advantage of a virus’s natural ability to enter mammalian cells efficiently. Once the delivery vehicle enters host cells, the encapsulated gene drives production of the missing or faulty protein. Clinicians use this approach for diseases such as muscular dystrophies, where mutations in the dystrophin gene cause progressive weakness and muscle-wasting, and cystic fibrosis, where mutations in the cystic fibrosis transmembrane conductance regulator gene impair lung function.

Non-viral Delivery Vehicles

Non-viral delivery vehicles, such as nanoparticles, can also deliver genes or gene editing systems in vivo. These particles use biocompatible materials such as polymers and lipids to carry genes or nucleic acids into cells.³ Researchers can customize a particle’s physiochemical properties to target the intended cell types better and evade immune responses. Non-viral particles are less efficient than viruses at entering cells but may have several other advantages. It’s likely that most patients are immunologically naïve to manufactured particles and lack pre-existing antibodies that may prevent successful uptake by cells. Non-viral particles may also be easier to manufacture and purify, making them more cost-effective to produce than viral vectors.⁴⁰

Recent gene therapy clinical trials using nanoparticles or lipid nanoparticles include the following^41-43:

• Reqorsa: contains a tumor suppressor gene encapsulated in a lipid nanoparticle for treatment of several types of lung cancer
• NTLA-2001: contains a gene editing system encapsulated in a lipid nanoparticle for treatment of hereditary transthyretin amyloidosis (a disease which deposits abnormal protein [amyloid] in organs)

Gene Editing Systems

Gene editing systems, which can be in vivo or ex vivo approaches, can provide precise, targeted gene modifications. Gene editors are engineered nucleases (enzymes) that produce double-stranded breaks at a specific target site. These breaks stimulate the cell’s natural DNA repair mechanisms, resulting in the repair of the break by homology-directed repair or non-homologous end joining. With these systems, target-specific DNA insertions, deletions, modifications, or replacements are all possible.^3,44

Many gene editing tools exist, including^3,44

• clustered regularly interspaced short palindromic repeats (CRISPR) Cas-associated nucleases
• transcription activator-like effector nucleases (TALENs)
• zinc-finger nucleases (ZFNs)
• meganucleases (MNs)

Gene editing ex vivo systems are a fast-growing area with many programs in development.⁴¹ In vivo gene editing is rife with challenges, especially those concerning delivery to the intended cells. Gene editing systems are an evolving cutting-edge platform reviewed in much more detail elsewhere.^3,44,45

Ex Vivo Gene Therapy

In ex vivo gene therapy, medical and/or laboratory personnel remove a patient’s cells from the body, genetically modify them (using viral vectors, non-viral delivery particles, or gene editing systems), and then re-introduce them into the patient. Alternatives to the use of a patient’s cells include genetically modified cell lines or cells from another donor. The use of autologous (patients’ own) cells avoids the need to find an immuno-compatible donor. Examples of systems include chimeric antigen receptor (CAR) T cells and engineered hematopoietic stem cells (HSC). CAR T cells are T lymphocytes that are modified to recognize tumor antigens, such as the B lymphocyte antigen Cluster of Differentiation-19 (CD-19) and B-cell maturation antigen (BCMA). Once genetically modified, the CAR T cells bind to cells expressing the tumor antigen and subsequently kill them. HSC have the ability for self-renewal and to differentiate into multiple cell lineages, therefore engineering them to correct genetic flaws such as enzyme deficiencies and hemoglobinopathies (inherited disorders affecting hemoglobin, the molecule that transports oxygen in the blood) makes them a promising approach.

Several products have received regulatory approval in the United States (U.S.)⁴⁶:

• Autologous CAR T cells^47-50
  • CD19-directed autologous T cell immunotherapies for the treatment of various types of B cell lymphomas include the following:
    • Yescarta (axicabtagene ciloleucel) – approved 2017
    • Kymriah (tisagenlecleucel) – approved 2017
    • Tecartus (brexucabtagene autoleucel) – approved 2020
    • Breyanzi (lisocabtagene maraleucel) – approved 2021
  • BCMA-directed autologous T cell immunotherapies for treatment of relapsed or refractory multiple myeloma include the following^51,52:
    • Abecma (idecabtagene vicleucel) – approved 2021
    • Carvykti (ciltacabtagene autoleucel) – approved 2022
• Autologous HSC-based gene therapies include the following^53-56:
  • Skysona (elivaldogene autotemcel) – slows neurological dysfunction in cerebral adrenoleukodystrophy by inducing synthesis of adrenoleukodystrophy protein – approved 2022
  • Zynteglo (betibeglogene autotemcel) – corrects β-thalassemia by adding functional copies of a modified β-globin gene into the patient’s HSC – approved 2022
  • Casgevy (exagamglogene autotemcel) – first FDA-approved CRISPR/Cas9 gene-editing therapeutic; corrects sickle cell disease (SCD) and transfusion-dependent β-thalassemia by adding functional copies of a modified β-globin gene into the patient’s HSC – approved 2023
  • Lyfgenia (lovotibeglogene autotemcel) – corrects SCD by adding functional copies of a modified β-globin gene into the patient’s HSC – approved 2023

CAR T cell gene therapy is an effective treatment for several types of cancers but comes with many challenges to patients, medical professionals, and pharmacy personnel. Product manufacturing, administration, and adverse effect mitigation are more complex than for many other treatment types. Medical, laboratory, and/or pharmacy personal carry out the following key steps at specialized medical centers^47-50:

1. Collect the patient’s white blood cells (leukapheresis)
2. Manufacture the CAR T cells in a laboratory with a typical time frame of two to four weeks
   • Patients may need to travel to and remain near the center while they wait for the cells
   • If the process fails, then personnel must repeat the cell collection and CAR T cell manufacturing
3. Administer chemotherapy to deplete the patient’s lymphocytes, two to 14 days prior to CAR T cell infusion, which makes room for the CAR T cells
4. Administer the CAR T cells to the patient by slow infusion to minimize infusion-related adverse reactions
5. Monitor the patient for at least four weeks, likely requiring the patient to stay at or near the treatment center

This therapy class carries Boxed Warnings:

• CD-19 directed therapies^47-50,57
  • Cytokine release syndrome (CRS): over-activation of the immune system with release of large amounts of cytokines (immune system proteins); this is a common, potentially life-threatening adverse event
  • Neurotoxicity: the mechanism by which this occurs is not well understood, but it is common and can be life-threatening

• BCMA-directed therapies^53,54
  • CRS and neurotoxicity as listed for CD-19 directed therapies
  • Hemophagocytic lymphohistiocytosis/macrophage activation syndrome: a hyperinflammatory immune response that can be life-threatening
  • Prolonged cytopenia (low numbers of blood cells) with bleeding and infection; can be life-threatening

Ex vivo gene therapy with autologous HSC is also a complex process with multiple challenges and toxicities.^53,54 For these therapies, medical personnel pretreat patients with granulocyte-colony stimulating factor to increase production and release of HSCs from bone marrow. Laboratory staff then collect and purify HSC from the patient’s blood. Staff may need to collect cells more than once to amass the required number of cells or in cases where manufacturing fails. Laboratory staff genetically modify the patient’s HSCs, typically with a lentiviral vector, to express the GOI. The cell manufacturing process can take up to three months. The modified cells require cryopreservation and liquid nitrogen storage conditions, adding to the process’s complexity.

When the cells are ready, medical personnel administer lymphocyte-depleting chemotherapy to the patient to remove HSCs that are making the faulty protein and to make room in the bone marrow for the genetically modified HSCs. Next, medical personnel infuse the thawed HSCs to the patient intravenously. After infusion, patients may need to stay at the medical center for several months for recovery and monitoring.

The prescribing information lists toxicities including prolonged cytopenias, serious infections, and an increased risk for lentiviral vector-mediated oncogenesis (development of a new cancer). It recommends patient monitoring for 15 years to life for hematologic malignancies. Skysona and Lyfgenia have Boxed Warnings for hematologic malignancy.^53,56

The overview above describes how the field of gene therapy is broad and encompasses multiple approaches. New and evolving approaches include non-viral delivery, gene editing, and ex vivo cell engineering; they have recently started to become available to patients. This is an exciting and hopeful time for patients with genetically-linked diseases, and it is likely that many more effective and approved therapies will be available soon.

AAV-BASED IN VIVO GENE THERAPY

Although researchers are developing viral vectors based on many types of viruses (e.g., adenovirus, gamma retrovirus, herpes simplex virus, lentivirus) adeno-associated virus (AAV) has emerged as the leading viral vector for in vivo gene therapy with five current regulatory approvals in the U.S. and many more programs under development.^41,46 Its favorable characteristics include not causing human disease, inability to replicate in the host without a helper virus, and ability to elicit a low host immune response relative to many other viruses. Since the expression cassette is typically maintained on an episome, scientists expect AAV-based vectors to have a low frequency of integration into the host genome, conferring a minimal risk of cancer.⁵⁸ In addition, at least one serotype of AAV (AAV9) can cross the blood-brain barrier making AAV a suitable choice for neurologic diseases without invasive local administration of the vectors into the brain. Researchers find AAV-based vectors to be attractive, versatile tools with suitable properties and utility for a wide variety of diseases.

AAV Biology

Naturally occurring AAVs are small, non-enveloped viruses that belong to the Parvovirus family. They have a linear single-stranded DNA genome of approximately 4.7 kilobases, including rep and cap genes that encode for replication and capsid proteins, respectively.³ Figure 1A illustrates the genomic structure of a wild type (naturally occurring) AAV.

In engineered AAVs used for gene therapy, developers remove the rep and cap genes and replace them with an expression cassette, as illustrated in Figure 1B. The expression cassette contains the GOI, regulatory elements (promoter/enhancer), and poly-adenosine tail (to stabilize the messenger RNA and facilitate translation into protein) flanked by inverted terminal repeats (ITRs; required for genome replication and packaging). The expression cassette replaces approximately 96% of the native genome.³

Figure 1. Genomic Structure of Wild Type AAV and Engineered AAV Vector

cap, capsid gene; GOI, gene of interest; ITR, inverted terminal repeat; rep, replicase gene.

AAV capsids in both the wild type AAV and engineered AAV vectors are composed of three viral proteins (VPs) named VP1, VP2, and VP3. Sixty molecules of these proteins, estimated to exist in a 1:1:10 ratio (VP1:VP2:VP3), assemble into each icosahedral capsid (Figure 2). The VP sequences overlap, with VP1 being 137 amino acids longer than VP2, which is 57 amino acids longer than VP3. Differences in VP3 sequences distinguish one AAV serotype from another.⁵⁹

Figure 2. Protein Structure of the AAV Capsid

VP, viral protein.

AAV exists as at least 11 natural serotypes, which share about 51% to 99% sequence identity in their VP3 protein.^60,61 Sequence comparisons between different AAV serotypes identified nine variable regions situated on the capsid surface. The specific amino acid sequences of these regions impact AAV binding to host cell receptors, thus determining cell- and tissue-specific tropism (affinity for a specific tissue or cell type). These variable sequences also function as binding sites (epitopes) for AAV-specific antibodies, thus influencing host immune responses.⁶²

An AAV serotype can be tropic to multiple cell types and tissues, and multiple serotypes can be tropic to the same cells/tissues. Researchers isolate serotypes from non-human primates and develop synthetic serotypes using protein engineering to alter tropisms and/or evade pre-existing AAV-specific antibodies.¹

PAUSE AND PONDER: If your child had a life-shortening genetic disorder and a new AAV9-based therapy became available, how would you feel if your child had a high anti-AAV9 neutralizing antibody titer that excluded them from clinical trials or treatment?

Neutralizing antibodies (NAbs) are naturally occurring antibodies that bind to AAV surface epitopes and block uptake into target cells. NAb binding to the AAV capsid can induce rapid clearance of the gene therapy by the patient’s immune system. Clinical personnel must screen patients for pre-existing Nabs. Trial protocols and prescribing instructions often exclude patients with NAbs specific for the gene therapy’s serotype.

Older children and adults tend to have a high prevalence of pre-existing antibodies against commonly circulating (wild type) AAV serotypes because of previous exposure to these viruses over the course of their lives. For example, studies showed that approximately 40% of humans have NAbs to AAV serotypes 5, 6, 7, 8, and 9.⁶³ Between 6% and 95% of individuals with hemophilia A and B have AAV-specific antibodies based on AAV serotype and assay used.⁶⁴ In addition, due to the high degree of conservation of capsid protein amino acid sequences, pre-existing antibodies to one serotype may cross-react with other serotypes, further limiting the availability of a therapy for a patient. Patients without NAbs prior to gene therapy will develop high titers of NAbs following vector administration, rendering them ineligible for additional rounds of gene therapy with the same vector or a cross-reactive serotype.^1,63-65

Vector Design

To create a vector for a particular disease, drug developers try to target an AAV vector to the specific cell or tissue type where gene correction is needed. The capsid serotype choice significantly influences this decision.

AAV2, AAV5, AAV6, AAV8, and AAV9 account for a majority of the ongoing AAV gene therapy clinical trials.^60,66 Commonly, AAV2 or AAV8 is the choice for ocular therapies; AAV2, AAV8, AAV5, and AAV9 are best for liver targeting; and AAV2 and AAV9 are preferred targeting muscle or the central nervous system. Some researchers are also using AAV serotypes isolated from rhesus monkeys, such as rh74.^60,67 As discussed, AAV9 can cross the blood-brain barrier, allowing IV administration of this serotype to target brain tissue while avoiding invasive intrathecal injections.^60,68 Therefore, AAV9 is a popular vector to treat many diseases affecting the nervous system.

Developers derive the capsids of AAV vectors either from natural AAV viruses or by engineering capsids through rational design, directed evolution, or computer-guided strategies. The impetus to develop AAV vectors with capsids from less common AAV serotypes or novel engineered AAV capsids serves to improve or modify tissue targeting and circumvent immune recognition.⁶⁹

Considerations for vector design include^61,62

• Choosing the best AAV serotype to target the intended cell type or tissue, using the desired route of administration
• Potential for tissue-specific promoters (e.g., targeting the muscle for muscular dystrophy or the liver for hemophilia) rather than constitutive promoters (that will drive gene expression in a wide range of tissues and perhaps result in off-target toxicity)
• Whether an enhancer is needed to boost expression in the target tissue to achieve desired therapeutic effect
• Modifying capsid epitopes to reduce recognition/response from the host immune system

Vector Production and Purification

Once developers choose the best capsid serotype and design the vector, they proceed to the manufacturing step. Gene therapies are complex biologics, and manufacturing and purification are of utmost importance for efficacy, safety, and product stability. Regulatory agencies require good manufacturing practice compliance.⁷⁰

Scientists commonly use mammalian cell lines to propagate engineered AAV vectors and then purify the vectors from cell lysates. Rigorous purification is essential. As the science of gene therapy matures, drug developers continuously improve purification methods. Contaminants from the engineered AAV cultivation process are a potential source of toxicity and can induce unwanted immune responses. Release testing of the final product in the U.S. must comply with a series of FDA-established requirements and predetermined specifications to determine product safety, purity, concentration, identity, potency, and stability.⁷¹ Characterization of the final product includes a determination of the viral genome (vg) titer, infectious titer (potency), product identity (AAV capsid and genome), and therapeutic gene identity (expression/activity).⁷²

Gene therapy developers follow these steps to manufacture AAV-based therapies^73-75:

1. Use conventional recombinant DNA technology in bacterial systems to produce the plasmids
2. Use mammalian cell lines, such as human embryonic kidney 293 cells, or insect cell lines, such as the baculovirus/insect cell system, to produce the vectors
3. Transfect (introduce genetic material into a cell) three plasmids into the cell line
   • a recombinant AAV plasmid containing the expression cassette (see Figure 1b)
   • an adenovirus-based helper plasmid supplying genes needed for virus replication
   • a plasmid containing the essential rep and cap genes needed for virus replication and capsid formation
4. Collect produced vectors from the cells and purify them. Complex purification processes require multiple methods that may be specific for each individual contaminant. Rigorous purification minimizes patient risk (see Sidebar). Examples of purification process steps (and techniques used) include the following:
   • Cells lysis to release the viral particles (detergent, mechanical stress, hypertonic shock, freeze-thawing)
   • Removal of nucleic acids (nuclease treatment), cell fragments and proteins, cell culture contaminants (centrifugation, filtration, chromatography)
   • Separation of full vector particles from empty particles (cesium chloride gradient ultracentrifugation or chromatography)
5. Formulate vectors after purification for administration to the patient. Formulation optimization prior to manufacture is not trivial. Some considerations are:^73-75
   • Formulation chemical composition must be optimized for intended route of administration
   • Formulation must be sterile and have low endotoxin concentration
   • Formulation must minimize product degradation to avoid aggregation and loss of efficacy due to product instability

SIDEBAR: Patient Safety: The Importance of Purification of AAV^69,74

• Non-vector sequences (including plasmid DNA, helper virus DNA, and DNA from the cell line used to produce the vector) can unintentionally be packaged into the capsids. AAV vector genomes can recombine with non-vector DNA, resulting in AAV vectors that contain chimeric (mixtures of DNA from different sources) sequences. The behavior of these unintended sequences is unpredictable in patients.
• Capsids containing the complete intended therapeutic gene, partial sequences, unintended chimeric sequences, or no genetic sequence material at all (empty capsids) are all possible outcomes of vector manufacturing. Administering a mixture of different forms of the therapeutic to patients may result in a partial effective dose, thus lowering potency and necessitating administration of higher overall numbers of AAV particles to achieve efficacy. The higher the number of AAV particles, the higher the risk of toxicities and immune system activation.

Toxicities/Adverse Events

Although AAV gene therapy is generally considered safe, multiple treatment-emergent serious adverse events have occurred following gene therapy administration to patients. These include severe hepatoxicity and thrombotic microangiopathy (TMA). Other potential toxicities and identified risks include myocarditis, neurotoxicity (dorsal root ganglion [DRG] degeneration), oncogenicity, and immunotoxicity.⁶⁹

In general, AAV vectors administered IV first travel to the liver. Liver enzyme increases denoting liver damage commonly occur and corticosteroid treatment is often effective. On occasion, acute serious liver injury, liver failure, and death have occurred. Although the mechanism is not completely understood, more severe liver injury appears to correlate with higher vector doses. Severe liver injury following AAV vector administration for spinal muscular atrophy (SMA) and X-linked myotubular myopathy genetic diseases has occurred.^69,76

TMA is a pathological condition characterized by formation of thrombi (clots) in small blood vessels, following endothelial cell injury. TMA’s clinical presentation often includes complement activation, cytokine release, thrombocytopenia (decreased platelets), and kidney injury/failure. Several cases of TMA have been reported in patients or clinical trial participants following AAV gene therapy for SMA and Duchenne muscular dystrophy (DMD). Treatments for affected patients included hemodialysis, platelet transfusion, and the complement inhibitor eculizumab. As with severe liver injury, TMA development appears to be related to high vector doses.^77,78

Several patients in DMD clinical trials have developed myocarditis, which was fatal in one patient. A hypothesis is that pre-existing inflammation in DMD patients’ muscles, including heart muscle, worsened upon local expression of dystrophin. Steroid treatment successfully resolved myocarditis in some cases.⁷⁹

DRG degeneration/toxicity is an identified potential risk of AAV gene therapy. DRGs are collections of sensory neurons that relay impulses from the periphery to the central nervous system. Researchers have observed AAV vector-induced DRG toxicity mainly in non-clinical studies with pigs and non-human primates. Direct administration of AAV into cerebral spinal fluid and high vector doses were contributing factors. Pathology studies have detected DRG degeneration in autopsy samples from humans who received AAV gene therapy. Scientists do not completely understand DRG toxicity’s underlying mechanisms in animals or the significance of clinical translation to humans.⁸⁰

Oncogenicity due to insertional mutagenesis (introducing foreign DNA into a person’s genome) is a potential risk of gene therapy. Studies in mice that received AAV vectors revealed integration events and hepatocellular carcinoma. Hepatocyte clonal expansion occurred in dogs following AAV vector administration although tumors were not found. AAV vector genome integration into chromosomes has been detected in humans, but so far AAV-associated oncogenesis has not been reported.⁵⁸ Due to the potential risk, regulators expect long-term monitoring after vector administration.^69,81

Although immune responses to wild type AAV are low relative to other viruses, AAV can engage all arms of the immune system and can do so robustly especially when high vector doses are administered. The immune response to AAV gene therapy is complex, and poorly understood, and can contribute to loss of efficacy and adverse effects. Local (rather than systemic) dosing, use of tissue specific promoters, and increasing the vector’s potency (increasing promoter strength/achieving higher levels of expression per AAV particle) may effectively minimize toxicity.^65,76,82,83

Immune responses to AAV gene therapy include the following^65,66,79,82:

• Innate immune responses: may lead to cytokine induction and/or activation of the complement cascade (e.g., toll-like receptor recognition and signaling in response to capsid proteins and nucleic acids)
• Humoral responses: antibody binding to AAV capsid may activate complement and immune cells and remove the vector from circulation before it can reach its target tissue. After gene therapy administration, the body generates new antibodies that can be specific for a capsid or transgene product.
• Cellular immune responses: may lead to inflammation and elimination of the cells producing the disease-correcting protein (e.g., activation/expansion of capsid- or transgene-product-specific cytotoxic T lymphocytes)

APPROVED IN VIVO GENE THERAPIES

As of December, 2023, the FDA approved eight in vivo gene therapies, five of which are AAV based.⁴⁶

Non-AAV-Based Therapies

Three of the FDA-approved in vivo gene therapies are non-AAV based; they are adenovirus (Ad)-based or herpes simplex virus (HSV)-based. None are systemically administered, but instead are administered locally to the skin or bladder.

Vyjuvek (beremagene geperpavec-svdt) is a live, replication deficient HSV type 1 (HSV-1) vector genetically modified to express human type VII collagen. Vyjuvek is indicated for wound treatment in patients with dystrophic epidermolysis bullosa with mutation(s) in the collagen type VII alpha 1 chain gene. Topical application to wounds induces production of the mature form of type VII collagen leading to wound healing.⁸⁴

Adstiladrin (nadofaragene firadenovec-vncg) is a non-replicating adenoviral serotype 5 vector genetically modified to produce human interferon α-2b (IFNα-2b). Adstiladrin is indicated for patients with high-risk Bacillus Calmette-Guérin-unresponsive non-muscle invasive bladder cancer with carcinoma in situ with or without papillary tumors. Intravesical (within the bladder) instillation results in local expression of IFNα-2b protein that is anticipated to have anti-tumor effects.⁸⁵

Imlygic (talimogene laherparepvec) is a live, attenuated HSV vector genetically modified to replicate within tumors and to produce the immune stimulatory protein granulocyte-macrophage colony-stimulating factor (GM-CSF). Imylgic is indicated for local treatment, by intralesional injection of unresectable cutaneous, subcutaneous, and nodal lesions in patients with melanoma recurrent after initial surgery. Imylgic causes tumor lysis followed by release of tumor-derived antigens, which together with virally derived GM-CSF may promote an antitumor immune response. However, the exact mechanism of action is unknown.⁸⁶

AAV-Based Therapies

Luxturna (voretigene neparvovec-rzyl) is a genetically modified non-replicating AAV2 expressing the human retinoid isomerohydrolase (RPE65) gene approved in 2017. It’s used to treat patients with confirmed biallelic RPE65 mutation-associated retinal dystrophy, a condition causing progressive retinal degeneration and dysfunction, which can lead to vision loss and blindness over time. Retinal pigment epithelial (RPE) cells produce RPE65. Mutations in this gene result in vision impairment, so Luxturna aims to correct these mutations. A clinician administers 1.5 x 10¹¹ vg of Luxturna subretinally (directly into the space beneath the retina) to each eye on separate days, at least six days apart. Patients typically take systemic oral corticosteroids starting three days before its administration and continuing with a tapering dose for 10 days. Preparation of Luxturna for administration is complex and includes thawing, dilution, and preparation of syringes for injection. Warnings and precautions include endophthalmitis (infection/inflammation of the inner structures of the eye), permanent decline in visual acuity, retinal abnormalities, increased ocular pressure, intraocular air bubble expansion, and cataracts. Immune responses to Luxturna were mild in clinical trials, perhaps due to corticosteroid administration.²³

Zolgensma (onasemnogene abeparvovec-xioi) is a genetically modified non-replicating AAV9 expressing the human survival motor neuron (SMN) protein. The FDA approved it in 2019 for treatment of patients younger than 2 years of age with spinal muscular atrophy (SMA) with bi-allelic mutations in the SMN1 gene. SMN protein is key for muscle development and movement and Zolgensma aims to correct these mutations to treat the disease. After Zolgensma is thawed, a clinician administers 1.1 × 10¹⁴ vg/kg of Zolgensma intravenously by slow infusion. Patients typically take systemic corticosteroids starting one day before Zolgensma administration and continuing for a total of 30 days. Serious adverse effects described in the Boxed Warning include acute liver injury and failure with fatal outcomes. Other warnings and precautions include systemic immune response, thrombocytopenia, TMA, and elevated troponin (a protein involved in muscle contraction).⁸⁷

Hemgenix (etranacogene dezaparvovec-drlb) is a genetically modified non-replicating AAV5 expressing human coagulation Factor IX (Padua variant), under control of a liver-specific promoter. Approved in 2022, it is employed to treat male patients with hemophilia B (congenital Factor IX deficiency). The Padua variant has an 8-fold higher specific activity relative to wild type Factor IX, and Hemgenix would ideally correct the disease by providing circulating Factor IX activity. Clinicians administer 2.0 × 10¹³ gc/kg of Hemgenix by slow intravenous infusion. Hemgenix is a refrigerated suspension that requires dilution into normal saline. The prescribing information recommends corticosteroids, not prophylactically, but only if the liver enzyme aspartate aminotransferase (AST) increases to twice the patient’s baseline value. Warnings and precautions include infusion reactions (including anaphylaxis), hepatotoxicity, and hepatocellular carcinogenicity (theoretical risk if vector DNA integrates into the host cell genome).⁸⁸

Elevidys (delandistrogene moxeparvovec-rokl) is a genetically modified non-replicating AAVrh74 (originally isolated from rhesus monkeys) expressing human micro-dystrophin. The FDA approved it in 2023 for treatment of ambulatory 4- to 5-year-old patients with DMD with a confirmed mutation in the dystrophin gene. The vector’s promoter/enhancer drives transgene expression predominantly in skeletal and cardiac muscle. The dystrophin protein’s micro version consists of selected domains of dystrophin that confer function to muscle cells. After Elevidys is thawed, clinicians administer 1.33 × 10¹⁴ vg/kg by intravenous infusion using a syringe infusion pump. The prescribing information recommends prophylactic corticosteroids following a complex administration and tapering schedule, which is further complicated in patients with DMD already on a corticosteroid regimen. Warnings and precautions include acute serious liver injury, immune-mediated myositis, and myocarditis.⁸⁹

Roctavian (valoctocogene roxaparvovec-rvox) is a genetically modified non-replicating AAV5 expressing the B-domain-deleted form of human coagulation Factor VIII (the smallest active form of Factor VIII) under control of a liver-specific promoter. Approved in 2023 for treatment of male patients with severe hemophilia A, clinicians administer Roctavian after thawing at 6.0 × 10¹³ vg/kg by intravenous infusion using a syringe infusion pump. The prescribing information recommends corticosteroids, not prophylactically, but only if liver enzymes increase to 1.5 times the patient’s baseline or to above the upper limit of normal. Warnings and precautions include infusion reactions (including anaphylaxis), hepatotoxicity, and thromboembolic events.⁹⁰

PAUSE AND PONDER: The prescribing information for Hemgenix and Roctavian do not recommend these gene therapies for women. Why?

A LOOK TO THE FUTURE

As discussed above, the FDA has approved five AAV-based gene therapies, Figure 3 illustrates disease indications being studied in AAV-based clinical trials including 90 ongoing, interventional studies.⁴¹

Figure 3. AAV-based gene therapy clinical trials for different disease indications as a percentage of all AAV-based clinical trials for therapies not yet approved for marketing⁴¹

The percentages of each disease indication were calculated based on the total of current AAV-based interventional clinical trials. Clinical trials were identified using the search terms gene therapy and adeno-associated (for condition/disease) and interventional (study type). Trials designated as recruiting, active – not yet recruiting, enrolling by invitation, and completed were included in the analysis (n = 73). Trials designated as terminated, suspended, or unknown status were not included. Data are current as of April 12, 2024.

Ophthalmology and neurology represent the highest percentage of forthcoming therapies. Within the ophthalmology category, multiple trials are underway for achromatopsia (color blindness), amaurosis (complete vision loss without visible eye damage; typically, an optic nerve disease), and retinitis pigmentosa (progressive vision loss due to retina deterioration), among others. The neurological disease category includes trials for Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, and others. Hematological and musculoskeletal clinical trials are highly focused on hemophilia and muscular dystrophies, respectively. The metabolic disease category is diverse containing a few trials each for numerous genetic diseases.⁴¹

Controlling the immune response to AAV vectors is one of the biggest hurdles for future therapeutics. The human immune system is complex and very efficient. Often, it has a back-up plan in case an organism escapes the first round of attack and usually a back-up plan for the back-up plan. It becomes impossible for an AAV vector (a virus in sheep’s clothing) to go unnoticed, especially when the doses are greater than 1 trillion vg/kg of body weight. Thus, a patient’s immune system will go into action starting shortly after receiving gene therapy and mount a multi-pronged attack.

New and improved strategies to control immune responses will greatly benefit patients. Researchers are diligently pursuing strategies (Table 2) that target the vector itself or immune system components.^91-93 Success will probably use a combinations of strategies.

Table 2. Strategies to Control or Circumvent Immune Responses^91-94

AAV, adeno-associated virus; IdeS, immunoglobulin G-degrading enzymes of Streptococcus pyogenes; IgG, immunoglobulin type G; NAb, neutralizing antibody.

Perfection of technologies to remove pre-existing NAbs would allow many more patients to be eligible for gene therapies. Curbing antibody production in the days following the gene therapy may boost efficacy, reduce toxicity, and allow patients to remain eligible for more than one round of AAV gene therapy if needed. Methods to control T cell-mediated responses would prevent destruction of the cells producing the transgene product, increasing the persistence of the curative gene expression.

Early clinical studies took a reactive approach to immune responses to AAV; they used medications such as corticosteroids following observation of increases in liver enzymes or signs of inflammation. More recently, efforts to identify drugs and combinations of drugs that will suppress the immune response prophylactically have accelerated^91,93:

• Corticosteroids (e.g., methylprednisolone): general anti-inflammatory
• Tacrolimus: inhibits T cell proliferation and differentiation
• Rituximab: depletes B cells to block antibody production
• Rapamycin (sirolimus): disrupts cytokine signaling resulting in inhibition of B cell and T cell activation
• Mycophenolate mofetil: inhibits B cell and T cell proliferation
• Eculizumab: complement inhibitor
• Hydroxychloroquine: inhibits toll-like receptor-9-mediated responses to viral DNA an antigen presentation

Researchers must investigate other key variables: the timing of immunosuppression initiation and the requisite treatment duration.

Many clinical trials have employed corticosteroids, but corticosteroids alone are insufficient to control immune responses. Regulators have approved the immunosuppressants listed above for indications other than gene therapy, and their safety profiles are known, making them logical choices to investigate for gene therapy indications. Future work must maximize safety and efficacy of AAV gene therapy while balancing adverse effects of the immunosuppressive regimens.⁹³

SUMMARY

Gene therapy is a relatively new, complex, and evolving field in medicine that offers hope to patients with previously incurable genetic diseases. The information presented here is intended to introduce a topic that pharmacists and pharmacy technicians may not have been previously exposed to, and perhaps inspire them to read more.

Download CE Activity

The University of Connecticut School of Pharmacy is accredited by the Accreditation Council for Pharmacy Education as a provider of continuing pharmacy education.

Pharmacists and Pharmacy Technicians are eligible to participate in this application-based activity and will receive 1.0 CE Hour  for completing the activity  (ACPE UAN 0009-9999-24-023-H01-P), passing the quiz with a grade of 70% or better, and completing an online evaluation. Statements of credit are available via the CPE Monitor online system and your participation will be recorded with CPE Monitor within 72 hours of submission.

• Drs. Micelli and Coughlin do not have any relationships with ineligible companies.

INTRODUCTION

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by persistent challenges in social interaction and communication, and restricted, repetitive patterns of behavior, interests, or activities. “Autism” has evolved from the early 1900s, when it was originally described as childhood schizophrenia in the first and second editions of the Diagnostic and Statistical Manual of Mental Disorders (DSM).¹ Autism was classified as an independent condition in the DSM-III in 1980. The DSM-IV in 1994 was the first to recognize autism as a spectrum with various distinct diagnoses. In 2013, the DSM-V consolidated autistic disorder, Asperger syndrome, pervasive developmental disorder-not otherwise specified, and childhood disintegrative disorder into the term known as autism spectrum disorder.²

The change in terminology has left many healthcare providers unsure about how to talk with and about people who have ASD. Researchers from the United Kingdom surveyed 654 English-speaking adults with ASD around the world to determine their preferences.³ Regardless of country, participants tended to favor the terms autism, autistic person, is autistic, neurological/brain difference, differences, challenges, difficulties, neurotypical people, and neurotypicals. Thus, the researchers were unable to find a universally accepted vernacular to talk about autism.³ Perhaps the best way to determine how to describe a patient with ASD is to ask how they themselves prefer to discuss their condition.

The term neurotypical is closely related to the term neurodiverse, and the SIDEBAR describes this relatively new term.

SIDEBAR: Neurodiversity^4-7

Medical insight and social changes in the perception of developmental disorders and neurodevelopmental trajectories, including the autism spectrum, have led to a changing vocabulary and a deeper appreciation of what is “normal.” Increasingly, we understand that human development is neurodiverse, meaning all individuals develop and behave differently. The concept of neurodiversity promotes the idea that people who have neurological limitations also have strengths, and that accommodating their differences as early in life as possible can be beneficial to the individual and to society at large. This is an empathetic, humanistic, tolerant approach.

It's now clear that people with ASD are often skilled in working with systems and finding patterns in complex data or material. This makes them good candidates to work in technology and manufacturing if an employer can accommodate an individual’s needs (e.g., quiet or dimly lit spaces, private or uncrowded work areas). People with dyslexia are often better than others at identifying peripheral or diffuse visual information or processing blurry visual scenes, making them excellent candidates for jobs that engage three-dimensional thinking (e.g., computer graphics, engineering, genetics, or molecular biology). Similarly, people with Williams syndrome (a rare genetic condition that affects physical features, development, and cardiovascular health), Down syndrome, and Prader-Willi syndrome (a rare genetic disorder causing weak muscles, poor feeding, and slow development in infants followed by constant hunger in childhood), tend to be more musical, more friendly, and more nurturing than others, respectively. Researchers have also connected specific strengths to other neurologic and intellectual disability diagnoses.

The origin of these strengths and limitations is probably linked to evolutionary adaptation. Being able to focus on patterns and systems, as seen in ASD, likely helped early humans in hunting and gathering societies. They were able to respond quickly to environmental stimuli and move in an appropriate direction when they identified potential prey. A famous quote comes from the autism activist Temple Grandin, who has autism. She has said, “Some guy with high functioning Asperger's developed the first stone spear; it wasn't developed by the social ones yakking around the campfire.”

The bottom line is that appreciating and respecting neurodiversity is kind and reasonable. Another quote from Temple Grandin explains it well: “Nature is cruel, but we don't have to be.”

Prevalence rates of ASD are reported to be approximately 1% worldwide (i.e., affecting 1 in 100 people), with comparable figures observed in samples of children and adults.⁸ Prevalence estimates have increased over time and vary greatly within and across socioeconomic and demographic groups. ASD is diagnosed four times more often in males than females.²

PAUSE AND PONDER: How many symptoms of autism spectrum disorder can you list before you continue reading?

Diagnosing ASD

According to the DSM-V, diagnosis of ASD requires persistent deficits in all three areas of social communication and interaction, in addition to at least two of four types of restricted, repetitive behaviors. Table 1 lists ASD’s core characteristics and symptoms.

Table 1. Symptoms of ASD²

Symptoms must be present in early development, cause clinically significant impairment in functioning, and cannot be attributable to intellectual disability or developmental delay. Since ASD is a spectrum, symptoms, severity, and treatment response vary widely among children and adults. Severity is categorized into three levels, described in Table 2.²

Table 2. Level of Severity of ASD²

Prognosis

One concern for many people with ASD and their caregivers is long-term prognosis. A recent systematic review looked at data from 16 small studies (two randomized, 14 non-randomized).⁹ Only three of the included studies enrolled more than 100 participants, limiting the ability to draw conclusions, but researchers found that early intervention improved children’s prognosis considerably.

Children who received intervention before 2 years of age were more likely to improve their cognition, social skills, and stereotyped behaviors than others. They needed less monitoring at school and were better able to function and integrate socially. These researchers noted that healthcare providers often fail to offer early intervention for four reasons⁹:

• Many healthcare providers are unfamiliar with the necessary screening, diagnosis, and intervention tools for ASD
• Early intervention involves many different strategies and is costly
• Involving parents in early intervention programs is difficult
• It’s difficult to recognize ASD’s signs in toddlers younger than 2 years

ASD CASE PRESENTATION

John, a 10-year-old boy with ASD, enters the community pharmacy with his mother to pick up his prescription for aripiprazole 10 mg tablets. His agitation due to the bright lights and loud chatter from other customers becomes immediately apparent to the pharmacy team. They notice several visible signs and hear auditory cues that indicate his discomfort and distress in the environment. John's body tenses up, with rigid posture and fidgety movements, and his hands are clenched tightly as he paces and rocks back and forth. His facial expressions—furrowed brows and widened eyes—convey distress, and his vocalizations include whimpers and "I don't like it here." Additionally, John exhibits repetitive behaviors such as hand-flapping and tapping his mother. He attempts to retreat from the situation by seeking refuge behind his mother. Understanding his sensitivity to sensory input, the pharmacy team must respond with patience, empathy, and sensitivity to ensure John feels supported and safe during his visit. In addition, John’s mother will appreciate empathy and accommodation.

Strategies for Support

Pharmacists can employ their medication expertise to help the healthcare team, families, and patients improve ASD management. As John’s description indicates, patients with ASD are often sensitive to sensory input like noise, light, and crowded environments.¹⁰ Pharmacists should recognize how a pharmacy setup can affect patients, potentially hindering communication. Offering to move to a quiet, dimly lit, private space (e.g., the consultation or vaccination area) for counseling helps accommodate sensory sensitivities.

When communicating with patients with ASD, pharmacists should consider the patient's level of autonomy and assess the need to interact primarily with the patient or the caregiver. Communication strategies can include using simplified language, direct communication, and patients' names to engage them and aid comprehension. Providing training to pharmacy staff on ASD can help pharmacists and technicians to serve these patients better.¹⁰

Putting Strategies in Action

The pharmacist, Keith, addresses John by name, acknowledges his discomfort, and minimizes distractions to create a more calming environment to put John at ease. For example, “Hello, John. I’m your pharmacist, and my name is Keith. It’s noisy out here. Would you like to move to a quieter room?” A technician familiar with the patient can also make this suggestion as soon as the patient arrives.

Keith also involves caregivers in the discussion, ensures they understand the medication instructions, and addresses any concerns they may have. He also reassures them of his availability for further assistance by saying, “I’m glad we talked about your medications today. If you have questions, you can stop in or call. I’m generally here Tuesday through Saturday, and my coworker Suzanne covers when I’m not here.”

By implementing these strategies, community pharmacists and technicians can improve the patient care experience for individuals with ASD.¹⁰

ASD TREATMENT

ASD is complex, and treatment often involves a multidisciplinary approach targeting various symptoms and challenges.¹¹ Although there is no outright “cure,” effective interventions may enhance functioning of children and adults with ASD. Pillars of treatment include behavioral therapies, speech and language therapy, occupational therapy, educational support, and sometimes medication management.

Behavioral therapy (e.g., applied behavioral analysis) involves creating a structured behavioral plan to improve adaptive skills and reduce inappropriate behaviors by studying affected individuals' functional difficulties systematically. Speech and language therapy is an integral part of ASD treatment for many children. Speech therapy targets difficulty with social communication and language development, which are some of ASD's core symptoms. Language therapy may employ visual supports like picture cards, augmentative and alternative communication devices, and teaching sign language (e.g., American Sign Language). Occupational therapy focuses on enhancing individuals' ability to participate in everyday activities and improve their quality of life. It typically addresses sensory processing issues, motor skill development, self-care skills, social interaction, and coping strategies, aiming to promote independence and function in various environments.¹¹

ASD treatment involves a multidisciplinary care team comprising professionals such as specialists, psychologists, pediatricians, paraprofessionals, and educators; ideally, team members collaborate to address individuals’ diverse needs and provide comprehensive support and intervention.¹¹

ASD Treatment Guidelines

Several treatment guidelines are available for ASD. The United Kingdom’s National Institute for Health and Care Excellence (NICE) has published ASD guidelines for both children under 19 years old and adults.^12,13 The Canadian Pharmacists Journal published ASD practice guidelines specifically for pharmacists. The latter guideline outlines strategies for effective communication and discusses how the community pharmacy team can create a welcoming environment for people with autism and their caregivers.¹⁰

Community pharmacists and technicians often encounter patients with diverse needs, including those with ASD. To provide optimal care, it's crucial to understand patients’ unique challenges and tailor services accordingly. Let's look at a case that highlights the importance of accommodating a patient with ASD in the pharmacy setting.

Pharmacologic Interventions

Pharmacotherapy for ASD primarily focuses on managing symptoms rather than directly targeting core features.⁸ However, many challenges exist in this area, including limited efficacy and evidence, adverse effects, individual variability, lack of targeted therapies, and long-term monitoring. Prescribers should consider that children with ASD often have heightened sensitivity to medication and are more prone to adverse reactions compared to neurotypical children. It is advisable to initiate pharmacologic treatment at lower doses and increase gradually based on response and tolerability. It's crucial to gather objective symptom measures from various sources both before and after intervention to assess treatment response accurately across different settings.⁸

Another major hurdle lies in addressing co-occurring disorders that often accompany ASD, such as attention-deficit/hyperactivity disorder (ADHD), anxiety, epilepsy, sleep disorders, and more. These additional conditions complicate treatment approaches, requiring tailored interventions to address unique needs. Very limited evidence supports the effectiveness of many medications used to manage ASD symptoms.¹⁴

The lack of medications specifically targeting core ASD symptoms and the limited efficacy data for various medications present barriers to identifying effective treatment strategies. Addressing these challenges requires a comprehensive approach that integrates pharmacologic interventions with behavioral, educational, and supportive therapies personalized to the individual's specific needs and circumstances. Additionally, ongoing research is crucial to advance our understanding of ASD and to develop more effective and targeted pharmacological treatments in the future.¹⁴

Most medications for ASD are used off-label and few United States Food and Drug Administration (FDA)-approved drugs are available for specific symptom management. Medications used may include atypical antipsychotics, stimulants, serotonergic drugs, alpha-2 adrenergic antagonists, anticonvulsants, and many others.¹⁴

Atypical Antipsychotics

Prescribers often use atypical antipsychotics for irritability associated with ASD. Currently, the FDA has approved only two medications for treatment of irritability associated with ASD: risperidone and aripiprazole.^15,16 Both are atypical (second generation) antipsychotics and exert effects through dopamine, 5-HT (serotonin), alpha-adrenergic, and histaminergic receptors in the brain.

Clinical trials have demonstrated effectiveness of these drugs in reducing irritability and, to a lesser extent, repetitive behaviors. They share similar safety profiles, with common adverse effects including fatigue, increased appetite, gastrointestinal symptoms, hyperprolactinemia, weight gain, and sedation. Less common adverse effects include restlessness and akathisia (inability to remain still). Serious adverse effects such as dyslipidemia, hyperglycemia, metabolic syndrome, and extrapyramidal symptoms (e.g., involuntary movements, muscle stiffness, tremors) or drug-induced movement disorders have also been reported, necessitating close clinical and laboratory monitoring.¹⁴

Risperidone is FDA-approved for treatment of ASD-associated irritability in children and adolescents aged 5 to 16 years.¹⁵ Studies have demonstrated that risperidone may effectively improve core symptoms of ASD, including communication, social interaction, and repetitive behaviors. Non-core symptoms such as aggression, tantrums, and self-injurious behaviors also improved based on various behavioral rating scales. Risperidone is generally well-tolerated and safe, with the most common adverse effect being mild, self-limiting weight gain. In one small study of 97 children treated with risperidone over a 6-month period, participants gained an average of 5.4 kg over 24 weeks. At baseline, 59 of the 97 children (60.8%) had normal weight status. By week 24, only 25 of the remaining 85 children (29.4%) maintained normal weights.¹⁷ In adults with ASD, adverse cognitive effects have not been observed in patients treated with risperidone for other psychiatric disorders. However, further exploration is needed regarding the efficacy of risperidone in adults with ASD.¹⁸

Aripiprazole is FDA-approved for treatment of ASD-associated irritability in pediatric patients 6 to 17 years old.¹⁶ Short-term studies have shown that aripiprazole can improve irritability, hyperactivity, and repetitive behaviors in children and adolescents with ASD compared to placebo. However, researchers have not observed improvement in lethargy or withdrawal symptoms. Aripiprazole use was associated with higher rates of movement disorders such as tremors and muscle rigidity. While aripiprazole may offer benefits, weight gain and neurological adverse effects like involuntary movements can limit its use. Regular monitoring of symptoms and adverse effects is recommended, and further research is needed to evaluate the long-term safety and effectiveness of aripiprazole in treating ASD.¹⁹

Other atypical antipsychotics occasionally used off-label for ASD include olanzapine, quetiapine, and ziprasidone. Providers generally avoid first generation antipsychotics due to the higher risk of movement-related adverse effects.¹⁴

Revisiting John’s Case

John's ASD is graded at Level 2—needing substantial support. He requires a range of support services tailored to his individual needs to thrive. For instance, John may benefit from behavioral interventions aimed at addressing his irritability and other behavioral challenges associated with his autism. These interventions could include strategies to manage his sensory sensitivities, develop coping skills, and enhance social communication. Additionally, providing John with structured routines and visual supports, such as clear schedules and visual cues, can help him navigate daily activities more effectively and reduce anxiety. Given his sensitivity to sensory stimuli, providing sensory accommodations like a quiet space or sensory tools (e.g., noise-canceling headphones) can aid in regulating his sensory experiences and minimizing agitation.

Moreover, John's prescription for aripiprazole indicates the need for medication management to address his irritability. Keeping in mind that John is 10 years old and in a period of rapid growth, it's crucial for the pharmacy team to monitor his height and weight regularly. The pharmacy team must inquire about any recent changes in his behavior or symptoms. The reason: the two atypical antipsychotics approved for irritability have similarities and differences that are critical to recognize, and with weight changes, the dose may require adjustments. The pharmacy team can support John's family, particularly his mother, with referral to resources such as parent training programs and support groups that can help them navigate the challenges associated with caring for a child with ASD. John can receive the assistance he needs to thrive and lead a fulfilling life with these comprehensive supports in place.

PAUSE AND PONDER: How many children with ASD receive care from your pharmacy? What behaviors do you see and hear?

Stimulants

Clinicians often prescribe stimulants to manage hyperactivity and inattention in ASD and co-existing ADHD. Two main classes of stimulants are commonly used in ADHD: amphetamines and methylphenidate derivatives.¹⁴ Prior to initiating stimulant therapy, clinicians must evaluate patients' medical and family histories and conduct a comprehensive physical exam focused on cardiovascular health. Ongoing monitoring for common adverse effects, such as appetite changes and sleep disturbances, is imperative, and it is essential to assess adolescent patients for risk of substance use or misuse before treatment initiation.

Amphetamine formulations include amphetamine-dextroamphetamine, dextroamphetamine, amphetamine sulfate, amphetamine, and lisdexamfetamine. Research suggests that amphetamines tend to be slightly more effective in reducing ADHD symptoms than methylphenidate, but they are also less tolerable. In a systematic review of data from more than 10,000 neurotypical individuals (i.e., children, adolescents, and adults without ASD), researchers found that amphetamines were more efficacious in reducing ADHD symptoms, although they were less well-tolerated than both placebo and methylphenidate in children and adolescents.²⁰

Methylphenidate products come in various formulations including immediate- and extended-release tablets or capsules, a transdermal patch, an extended-release liquid, and orally disintegrating tablets. Short-term treatment with methylphenidate has shown some benefit in improving hyperactivity, inattention, and other ADHD symptoms in children with ASD, but studies are small. The largest has enrolled just 66 children.²¹ Nonetheless, no evidence showed improvement in core ASD symptoms or social interaction. Additionally, while some children with ASD responded positively to methylphenidate, a significant number experienced adverse effects such as irritability, repetitive behaviors, insomnia, and reduced appetite.¹⁴

Alpha-2 Adrenergic Agonists

Alpha-2 adrenergic agonists—including guanfacine and clonidine—are commonly used in ADHD management for younger children. Evidence exists regarding the off-label use of alpha-2 adrenergic agonists in alleviating some symptoms of ASD, so some providers use them off label for this condition. Clinicians commonly prescribe these for children younger than 5 years old with ADHD or hyperarousal (intense, rapid, and often overwhelming emotional responses). These medications are also useful in cases when patients have poor responses to stimulants or selective norepinephrine reuptake inhibitors (e.g., atomoxetine) or when patients have significant co-occurring conditions like sleep issues that preclude stimulant use.

Research on the use of alpha-2-adrenergic agonists in ASD is limited to a few small studies.¹⁴ Guanfacine has been shown to be safe and effective in managing hyperactivity and impulsiveness in children with ASD. Common adverse effects of guanfacine include sedation, constipation, irritability, and aggression, but they are generally better tolerated than stimulant medications.¹⁴

PAUSE AND PONDER: What kinds of questions should you ask caregivers when they present a new prescription for a patient with ASD? What information do they need to know (but might not think about)?

Other Medications with Limited Supporting Data

Several medications have been explored with limited evidence in ASD management. These include various serotonergic medications (selective norepinephrine receptor inhibitors [SNRIs] and selective serotonin reuptake inhibitors [SSRIs]), anticonvulsants, gabapentin, and trazodone.

Researchers have looked at SNRIs and SSRIs to treat difficult behaviors in ASD. Venlafaxine has been proven beneficial as an adjunct treatment for self-injurious behaviors, aggression, and ADHD symptoms in children and adults with ASD, particularly when administered at doses lower than those typically used for depression.²² Conversely, duloxetine (also an SNRI) did not demonstrate any additional advantages in addressing comorbid symptoms and behaviors associated with ASD when compared to alternative antidepressants.²²

SSRIs can be helpful in patients with comorbid anxiety, which is very common with ASD. However, clinical trials assessing the SSRIs citalopram and fluoxetine found them to have low tolerability and limited effectiveness in addressing repetitive behaviors.²³

Anticonvulsants, also known as antiepileptic drugs, are sometimes used off-label in the treatment of certain ASD symptoms. While these medications are primarily indicated for seizure management, they may also be prescribed to address co-occurring conditions such as epilepsy, aggression, irritability, and repetitive behaviors in individuals with ASD. Examples of anticonvulsants studied or used in ASD treatment include valproic acid (valproate), lamotrigine, levetiracetam, and topiramate.¹⁴ However, the evidence supporting anticonvulsants' efficacy in treating ASD symptoms remains limited, and healthcare professionals should consider the use of these drugs carefully and monitor closely for potential adverse effects and individual variability in response. ¹⁴

Limited research suggests that carbamazepine, oxcarbazepine, levetiracetam, and topiramate may exacerbate hyperactivity, mood disturbances, psychotic symptoms, and other psychiatric or behavioral issues in individuals with ASD.²⁴ These effects appear most common with levetiracetam. Evidence is inconclusive on the role of anticonvulsants in ASD in the absence of epilepsy, but there always remains potential for specific cases. Further research is needed to better understand the safety and effectiveness of anticonvulsants in ASD treatment and to identify subgroups of individuals who may benefit most from this approach.

Off-label use of gabapentin and trazodone for ASD presents significant patient safety concerns. Despite lacking FDA approval for this indication, these drugs are increasingly prescribed, leading to adverse drug reactions.²⁵ Gabapentin is often used off-label to address anxiety and occasionally behavioral problems. Gabapentin can also cause central nervous system depression. Its use in ASD is poorly studied, with one study that enrolled just 23 children (mean age 7.2) with various neurologic diagnoses finding that 78% of children had improved sleep at doses of 5 mg/kg 30 to 40 minutes before bedtime.²⁶ However, further research is required to substantiate these findings.²⁶

Very limited evidence supports using trazodone to manage irritability, but many individuals with ASD still use it, as sleep disturbances are very common. Safety considerations exist, as trazodone poses risks of overdose-related complications, including arrhythmias, respiratory arrest, coma, and the rare but serious condition of priapism. Overprescribing of these medications is increasing and not backed by evidence, especially in ASD. Cautious prescribing practices and thorough patient education are needed to ensure the safety and well-being of individuals with ASD using gabapentin and trazodone.

As mentioned, sleep problems are common in children with ASD. In fact, between 40% and 80% of children with ASD develop them.²⁷ For many children, insomnia is the problem, but other children develop parasomnias (e.g., sleep talking, sleepwalking, sleep terrors), or circadian rhythm sleep-wake disorders. Prescribers have various options available to treat insomnia, but in autism, the largest body of work describes the use of melatonin to improve sleep onset and maintenance. Research suggests using lowest doses (1 to 2 mg) and titrating upward gradually.^28,29

John Develops Insomnia

Once again, John and his mother visit the pharmacy to fill a prescription. His mom says that although they have a very structured schedule and John's regular bedtime is 9:00 PM, John has difficulty falling asleep and is often awake and moving around in his room for long periods of time. His mom says, “Recently, it feels like he's awake all night long and his symptoms are worse when he doesn't get enough sleep. It's a vicious cycle.” She looks exhausted; a key issue when children with ASD develop sleep disorders is that the entire family often loses sleep.

Mom also reports that her primary care provider has counseled her on ways to help John get to sleep, including discouraging behaviors that interrupt sleep, using positive reinforcement when John is actively trying to sleep, and employing relaxation techniques. She says that she moved his bedtime to 11:00 PM and he has been a little bit sleepier. Her plan is to move John’s bedtime 15 minutes earlier each week. Regardless, John and his entire family still need additional help with this issue. John’s mother indicates that the prescriber told her to speak with the pharmacist so she can find the most reliable melatonin product. That’s a prudent recommendation, since many supplements are mislabeled or unreliable.²⁸

PHARMACY IMPLICATIONS

Pharmacists, pharmacy technicians, and the community pharmacy setting serve as essential components of healthcare for patients with ASD and their families. The pharmacy team can address concerns, provide encouragement, and ensure parents and caregivers feel equipped to manage medication administration effectively. Pharmacists can provide counseling and technicians can offer support to help caregivers confidently manage medication regimens for patients with ASD, ensuring better adherence and improved healthcare outcomes. Table 3 lists important reminders for the pharmacy team.

Table 3. Best Interventions for Children with ASD at the Pharmacy^10,30,31

• Anticipate that you will provide care for patients with ASD and identify a calm area where you can council without noise or distraction
• Accept that some parents do not want to take any more time in a store (pharmacy or not) than they must, and try to accommodate them
• At every visit, ask if anything has changed since the last visit and record an updated height and weight (especially if the patient is on a medication dosed by weight)
• If parents struggle with administering medications to children with ASD
  • Provide tailored support and education, offer clear and concise medication instructions using visual aids and use simplified language
  • Recommend dosage forms like liquids or chewable tablets to ease administration challenges
  • Offer customized packaging options such as unit-dose blister packs or pre-filled syringes proactively to simplify dosing and organization
  • Help parents and caregivers create visual medication schedules or reward systems to reduce anxiety during medication administration
• Encourage parents and caregivers to keep a diary of symptoms so they can monitor the patient’s response to newly prescribed medications
• Remember that stimulants, gabapentin, and trazodone have been linked to abuse and all families need to be reminded to store these drugs securely
  • In some states, gabapentin is a controlled substance

Pharmacists are a valuable drug information resource and adept at assessing existing evidence to help patients with ASD and their families make well-informed decisions. Pharmacy involvement is invaluable in providing optimal care and support for this patient population.

The Future of Treating ASD

The future of pharmacologic treatment for ASD holds promise but also faces significant challenges. Continued research into ASD's underlying neurobiologic mechanisms may lead to the development of more targeted interventions tailored to address specific symptoms and subtypes of the disorder. Additionally, advancements in genetic testing and biomarker identification could enable personalized treatment approaches, optimizing efficacy while minimizing adverse effects.

Large-scale clinical trials and collaborative research efforts to evaluate treatment effectiveness comprehensively are needed. Ultimately, the future of medication therapy for ASD will depend on the ability to integrate advancements with a nuanced understanding of individual differences and needs of patients with ASD.¹⁴

CONCLUSION

While progress has been made in understanding and treating ASD, significant challenges remain. A comprehensive approach combining behavioral interventions, therapies, and pharmacotherapy tailored to individual needs is essential for improving outcomes in individuals with ASD. Continued research efforts are necessary to develop more effective and evidence-based treatments for this complex disorder. Temple Grandin explains it well: “A treatment method or an educational method that will work for one child may not work for another child. The one common denominator for all of the young children is that early intervention does work, and it seems to improve the prognosis.”

Download CE Activity